Bioactive components conjugated to substrates of microneedle arrays

ABSTRACT

Microneedle arrays and methods of forming the same can include one or more bioactive components bonded to a biocompatible material such that the one or more bioactive components are cleavable in vivo to release the bioactive component from the biocompatible material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/135,052, filed Mar. 18, 2015; and U.S. Provisional Application No.62/135,643, filed Mar. 19, 2015; each of which is herein incorporated byreference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number NIHP50 CA121973 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

The disclosure pertains to systems and methods for transdermal drugdelivery, and, in particular, to systems and methods for making andusing dissolvable microneedle arrays.

BACKGROUND

The remarkable physical barrier function of the skin poses a significantchallenge to transdermal drug delivery. To address this challenge, avariety of microneedle-array based drug delivery devices have beendeveloped. For example, one conventional method employs solid or hollowmicroneedles arrays with no active component. Such microneedle arrayscan pre-condition the skin by piercing the stratum corneum and the upperlayer of epidermis to enhance percutaneous drug penetration prior totopical application of a biologic-carrier or a traditional patch. Thismethod has been shown to significantly increase the skin's permeability;however, this method provides only limited ability to control the dosageand quantity of delivered drugs or vaccine.

Another conventional method uses solid microneedles that aresurface-coated with a drug. Although this method provides somewhatbetter dosage control, it greatly limits the quantity of drug delivered.This shortcoming has limited the widespread application of this approachand precludes, for example, the simultaneous delivery of optimalquantities of combinations of antigens and/or adjuvant in vaccineapplications.

Another conventional method involves using hollow microneedles attachedto a reservoir of biologics. The syringe needle-type characteristics ofthese arrays can significantly increase the speed and precision ofdelivery, as well as the quantity of the delivered cargo. However,complex fabrication procedures and specialized application settingslimit the applicability of such reservoir-based microneedle arrays.

Yet another conventional method involves using solid microneedle arraysthat are biodegradable and dissolvable. Current fabrication approachesfor dissolvable polymer-based microneedles generally use microcastingprocesses. However, such conventional processes are limited in theactive components that can be embedded into the array and are alsowasteful in that they require that the active components be homogenouslyembedded in the microneedles and their support structures.

Accordingly, although transdermal delivery of biologics usingmicroneedle-array based devices offers attractive theoretical advantagesover prevailing oral and needle-based drug delivery methods,considerable practical limitations exist with conventional deliverysystems and methods.

SUMMARY

The systems and methods disclosed herein include cutaneous deliveryplatforms based on dissolvable microneedle arrays that can provideefficient, precise, and reproducible delivery of biologically activemolecules to human skin. The microneedle array delivery platforms can beused to deliver a broad range of bioactive components to a patient.

In one embodiment, a dissolvable microneedle array for transdermalinsertion into a patient is provided that includes a substratecomprising a biocompatible material that forms base portion and aplurality of microneedles extending from the base portion and one ormore bioactive components conjugated to the biocompatible material. Theone or more bioactive components are cleavable in vivo to release thebioactive component from the biocompatible material, or the bioactivecomponent retains function when conjugated to the biocompatiblematerial.

The one or more bioactive components can be covalently bonded to thebiocompatible material, such as by a disulfide bond. The microneedlearray of claim 1, wherein the biocompatible material can becarboxymethylcellulose. The one or more bioactive components can becleavable in vivo by an enzyme, and/or in response to pH, temperature,or both. The one or more bioactive components can be the same ordifferent bioactive components, and one, or both, of the bioactivecomponents can be conjugated to the biocompatible material. The at leasttwo different bioactive components are selected from the groupconsisting of a chemotherapeutic agent, an adjuvant, and a chemoattractant for a cancer chemo immunotherapy application. In otherembodiments, the bioactive component can include an antigen and anadjuvant for a vaccine application, and/or can include at least oneviral vector, which can comprise in some cases an adenovector.

In some examples, the one or more bioactive components compriseDoxorubicin, such as in an amount from about 1 to 1000 μg forchemotherapy. In some examples, the microneedle arrays can provide theone or more bioactive components in a higher concentration in theplurality of microneedles than in the base portion. In other examples,substantially all of the one or more bioactive components are located inthe plurality of microneedles so that the base portion is substantiallyformed without any bioactive components contained therein. The one ormore bioactive components can be locally concentrated in the pluralityof microneedles so that the one or more bioactive components aregenerally present only in an upper half of respective microneedles inthe microneedle array. Each microneedle can include a plurality oflayers of the biocompatible material.

A method of fabricating a microneedle array is also provide. The methodincludes applying a first solution of a dissoluble biocompatiblematerial having one or more bioactive components conjugated to thedissoluble biocompatible material therein to a microneedle arrayproduction mold; applying a second solution of the dissolublebiocompatible material that does not contain one or more activecomponents to the microneedle array production mold; and drying thefirst and second solutions to form a solid microneedle array thatcomprises a base portion and a plurality of microneedles that extendfrom the base portion. The one or more active components can besubstantially concentrated in the plurality of microneedles, and the oneor more bioactive components can be covalently bonded to the dissolublebiocompatible material such that the conjugated bioactive components arecleavable in vivo to release the bioactive component from thebiocompatible material.

In another embodiment, a dissolvable microneedle array for transdermalinsertion into a patient is provided. The array includes a substratecomprising a biocompatible material that forms base portion and aplurality of microneedles extending from the base portion, and a firstbioactive component conjugated to the biocompatible material. Thebioactive component is Doxorubicin and the Doxorubicin is cleavable invivo to release it from the biocompatible material, or the Doxorubicinretains function when conjugated to the biocompatible material.

In some embodiments, the Doxorubicin is covalently bonded to thebiocompatible material (e.g., CMC), such as by a disulfide bond. TheDoxorubicin can be cleavable in vivo by an enzyme, and/or cleavable invivo in response to pH, temperature, or both. The amount of Doxorubicinranges from about 1 to 1000 μg for chemotherapy.

In other embodiments, the array also includes a second bioactivecomponent, such as one selected from the group consisting of Poly-IC orPoly-ICLC. The second bioactive component can also conjugated to thebiocompatible material or it can be mixed into the biocompatiblematerial.

The foregoing and other objects, features, and advantages of thedisclosed embodiments will become more apparent from the followingdetailed description, which proceeds with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary microneedles and their dimensions.

FIG. 2 illustrates an exemplary microneedle array and its dimensions.

FIGS. 3A and 3B illustrate exemplary microneedles with tip-loaded activecomponents.

FIGS. 4A and 4B illustrate exemplary microneedles with tip-loaded activecomponents.

FIGS. 5A and 5B illustrate exemplary microneedles with tip-loaded activecomponents.

FIGS. 6A and 6B illustrate exemplary microneedles with tip-loaded activecomponents.

FIG. 7 illustrates a miniature precision-micromilling system used forfabricating microneedle mastermolds.

FIG. 8 is an SEM image of a micromilled mastermold with pyramidalneedles.

FIG. 9 is an SEM image of a pyramidal production mold.

FIG. 10 is an SEM image of an enlarged segment of the production mold,illustrating a pyramidal needle molding well in the center of the image.

FIGS. 11A-11D illustrate exemplary CMC-solids and embedded activecomponents.

FIGS. 12A-12B illustrate exemplary CMC-solids and embedded activecomponents.

FIG. 13 is a schematic illustration of exemplary vertical multi-layereddeposition structures and methods of fabricating the same.

FIG. 14 is a schematic illustration of exemplary microneedle arraysfabricated using layering and spatial distribution techniques ofembedded active components.

FIG. 15 is a schematic illustration of exemplary microneedle arraysfabricated in a spatially controlled manner.

FIG. 16A is an SEM image of a plurality of pyramidal-type moldedmicroneedles.

FIG. 16B is an SEM image of a single pyramidal-type molded microneedle.

FIG. 17 is an SEM image of a pillar type molded microneedle.

FIG. 18 is a micrograph of pyramidal type molded microneedles.

FIG. 19 is a micrograph of pillar type molded microneedles.

FIG. 20 illustrates various microneedle geometries that can be formedusing micromilled mastermolds or by direct micromilling of a block ofmaterial.

FIG. 21 illustrates a test apparatus for performing failure and piercingtests.

FIG. 22 illustrates force-displacement curves for pillar typemicroneedles (left) and pyramidal type microneedles (right).

FIG. 23 illustrates a finite elements model of microneedle deflectionsfor pillar type microneedles (left) and pyramidal type microneedles(right).

FIGS. 24A-24F show various stereo micrographs of the penetration ofpyramidal (A, C, E) and pillar (B, D, F) type microneedles in skinexplants.

FIGS. 25A-25C illustrate the effectiveness of microneedle arrays inpenetrating skin explants.

FIGS. 26A and 26B illustrate in vivo delivery of particulates to theskin draining lymph nodes of microneedle array immunized mice.

FIG. 27 is a bar graph showing immunogenicity of microneedle deliveredmodel antigens.

FIG. 28 is a bar graph showing the stability of the active cargo ofCMC-microneedle arrays in storage.

FIGS. 29A and 29B show induction of apoptosis in epidermal cells thathave been delivered Cytoxan® (cyclophosphamide) through a microneedlearray.

FIG. 30 illustrates a microneedle geometry that can be formed by directmicromilling of a block of material.

FIG. 31 is a stereo microscopic image of a direct-fabricated solidCMC-microneedle array.

FIG. 32 is a stereo microscopic image of a portion of the microneedlearray of FIG. 31.

FIG. 33 is a schematic cross-sectional view of a casting-mold assemblyfor creating a block or sheet of material for direct micromilling.

FIG. 34 is a schematic cross-sectional view of a drying apparatus thatcan be used to dry a block or sheet of material for direct micromilling.

FIG. 35 is a flow cytometry analysis of GFP expressing target 293Tcells.

FIG. 36 illustrates the stability of microneedle embedded viruses aftera number of days in storage.

FIG. 37 illustrates the expression and immunogenicity of microneedlearray delivered adenovectors.

FIG. 38 shows a chemical schematic of the Doxorubicin-S—S-CMC construct.

FIG. 39 is a schematic of preparing of NH₂-cellulose for solid supportusing epichlorohdrin and ammonium hydroxide.

FIG. 40 shows Doxorubicin S—S cross-linked to cellulose support.

FIG. 41 shows Doxorubicin-S—S-cellulose and cellulose sample from shamreaction prepared for cleavage and elution in a column.

FIG. 42 shows a two-step ammination of CMC in alkaline environment usingepichlorohydrinand ammonium hydroxide.

DETAILED DESCRIPTION

The following description is exemplary in nature and is not intended tolimit the scope, applicability, or configuration of the disclosedembodiments in any way. Various changes to the described embodiment maybe made in the function and arrangement of the elements described hereinwithout departing from the scope of the disclosure.

As used in this application and in the claims, the singular forms “a,”“an,” and “the” include the plural forms unless the context clearlydictates otherwise. Additionally, the term “includes” means “comprises.”As used herein, the terms “biologic,” “active component,” “bioactivecomponent,” “bioactive material,” or “cargo” refer to pharmaceuticallyactive agents, such as analgesic agents, anesthetic agents,anti-asthmatic agents, antibiotics, anti-depressant agents, antidiabeticagents, anti-fungal agents, anti-hypertensive agents, anti-inflammatoryagents, anti-neoplastic agents, anxiolytic agents, enzymatically activeagents, nucleic acid constructs, immunostimulating agents,immunosuppressive agents, vaccines, and the like. The bioactive materialcan comprise dissoluble materials, insoluble but dispersible materials,natural or formulated macro, micro and nano particulates, and/ormixtures of two or more of dissoluble, dispersible insoluble materialsand natural and/or formulated macro, micro and nano particulates.

As used herein, the term “pre-formed” means that a structure or elementis made, constructed, and/or formed into a particular shape orconfiguration prior to use. Accordingly, the shape or configuration of apre-formed microneedle array is the shape or configuration of thatmicroneedle array prior to insertion of one or more of the microneedlesof the microneedle array into the patient.

As used herein, the term “conjugate” means two or more moieties directlyor indirectly coupled together. Two entities are conjugated when underphysiological conditions of pH, ionic strength and osmotic potential,the majority of the entities are associated with each other atequilibrium, such as due to the presence of a convalent bond. Covalentlinkage may be by any of a variety of chemical linking and cross-linkingagents including, for example, homobifunctional or heterobifunctionalcrosslinking reagents, many of which are commercially available (see,e.g., Pierce Chemical Co. or Sigma Chemical Co.). Linking orcrosslinking can be achieved by any of a variety of chemistries wellknown in the art including, for example, activated polyethylene glycols,aldehydes, isocyanates, maleimides and the like. Linking orcross-linking can also be achieved using physical methods, such asirradiation, for example gamma irradiation or ultraviolet (UV)irradiation. For example, a first moiety may be covalently ornoncovalently (e.g., electrostatically) coupled to a second moiety.Indirect attachment is possible, such as by using a “linker” (a moleculeor group of atoms positioned between two moieties).

As used herein, “cancer” means a malignant neoplasm that has undergonecharacteristic anaplasia with loss of differentiation, increase rate ofgrowth, invasion of surrounding tissue, and is capable of metastasis.For example, thyroid cancer is a malignant neoplasm that arises in orfrom thyroid tissue, and breast cancer is a malignant neoplasm thatarises in or from breast tissue (such as a ductal carcinoma). Residualcancer is cancer that remains in a subject after any form of treatmentgiven to the subject to reduce or eradicate thyroid cancer. Metastaticcancer is a cancer at one or more sites in the body other than the siteof origin of the original (primary) cancer from which the metastaticcancer is derived. Cancer includes, but is not limited to, solid tumors.

As used herein, “tumor” means an abnormal growth of cells, which can bebenign or malignant. Cancer is a malignant tumor, which is characterizedby abnormal or uncontrolled cell growth. Other features often associatedwith malignancy include metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels and suppression or aggravation ofinflammatory or immunological response, invasion of surrounding ordistant tissues or organs, such as lymph nodes, etc. “Metastaticdisease” refers to cancer cells that have left the original tumor siteand migrate to other parts of the body for example via the bloodstreamor lymph system. The amount of a tumor in an individual is the “tumorburden” which can be measured as the number, volume, or weight of thetumor. A tumor that does not metastasize is referred to as “benign.” Atumor that invades the surrounding tissue and/or can metastasize isreferred to as “malignant.”

As used herein, “chemotherapeutic agents” means any chemical agent withtherapeutic usefulness in the treatment of diseases characterized byabnormal cell growth. Such diseases include tumors, neoplasms, andcancer as well as diseases characterized by hyperplastic growth such aspsoriasis. In one embodiment, a chemotherapeutic agent is an agent ofuse in treating neoplasms such as solid tumors. In one embodiment, achemotherapeutic agent is radioactive molecule. One of skill in the artcan readily identify a chemotherapeutic agent of use (e.g. see Slapakand Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., © 2000Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer D S, Knobf M F, Durivage H J (eds): The Cancer ChemotherapyHandbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeuticagents include those known by those skilled in the art, including butnot limited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide,antimetabolites (such as Fludarabine), antineoplastics (such asEtoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin,cis-platinum and the taxanes, such as taxol. Rapamycin has also beenused as a chemotherapeutic.

As used herein, “therapeutically effective dose” means a dose sufficientto prevent advancement, or to cause regression of a disease, or which iscapable of relieving symptoms caused by a disease, such as pain.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Although the operations of exemplary embodiments of the disclosed methodmay be described in a particular, sequential order for convenientpresentation, it should be understood that disclosed embodiments canencompass an order of operations other than the particular, sequentialorder disclosed. For example, operations described sequentially may insome cases be rearranged or performed concurrently. Further,descriptions and disclosures provided in association with one particularembodiment are not limited to that embodiment, and may be applied to anyembodiment disclosed.

Moreover, for the sake of simplicity, the attached figures may not showthe various ways (readily discernable, based on this disclosure, by oneof ordinary skill in the art) in which the disclosed system, method, andapparatus can be used in combination with other systems, methods, andapparatuses. Additionally, the description sometimes uses terms such as“produce” and “provide” to describe the disclosed method. These termsare high-level abstractions of the actual operations that can beperformed. The actual operations that correspond to these terms can varydepending on the particular implementation and are, based on thisdisclosure, readily discernible by one of ordinary skill in the art.

Tip-Loaded Microneedle Arrays

Dissolvable microneedle arrays enable efficient and safe drug andvaccine delivery to the skin and mucosal surfaces. However, inefficientdrug delivery can result from the homogenous nature of conventionalmicroneedle array fabrication. Although the drugs or other cargo that isto be delivered to the patient are generally incorporated into theentire microneedle array matrix, in practice only the microneedles enterthe skin and therefore, only cargo contained in the volume of theindividual needles is deliverable. Accordingly, the vast majority of thedrugs or other cargo that is localized in the non-needle components(e.g., the supporting structure of the array) is never delivered to thepatient and is generally discarded as waste.

FIGS. 1 and 2 illustrate exemplary dimensions of microneedles andmicroneedle arrays. Based on the illustrative sizes shown in FIGS. 1 and2, a microneedle array that comprises an active component homogenouslydistributed throughout the array exhibits active component waste ofgreater than 40 percent. For example, if the entire area of the array is61 mm² and the microneedle array area is 36 mm², then the percentutilization of the active component is less than 60 percent. Althoughthe dimensions reflected in FIGS. 1 and 2 illustrate a particular sizearray and shape of microneedles, it should be understood that similarwaste is present in any other size microneedle array in which the activecomponent is homogenously distributed throughout the array, regardlessof the size of the array or the shape of the microneedles involved.

The systems and methods described herein provide novel microneedle arrayfabrication technology that utilizes a fully-dissolvable microneedlearray substrate and unique microneedle geometries that enable effectivedelivery of a broad range of active components, including a broad rangeof protein and/or small molecule medicines and vaccines.

As described in more detail herein, in some embodiments, this technologycan also uniquely enable the simultaneous co-delivery of multiplechemically distinct agents for polyfunctional drug delivery. Examples ofthe utility of these devices include, for example, (1) simultaneousdelivery of multiple antigens and adjuvants to generate a polyvalentimmune response relevant to infectious disease prevention and cancertherapy, (2) co-delivery of chemotherapeutic agents, immune stimulators,adjuvants, and antigens to enable simultaneous adjunct tumor therapies,and (3) localized skin delivery of multiple therapeutic agents withoutsystemic exposure for the treatment of a wide variety of skin diseases.

In some embodiments, the systems and method disclosed herein relate to anovel fabrication technology that enables various active components tobe incorporated into the needle tips. Thus, by localizing the activecomponents in this manner, the remainder of the microneedle array volumecan be prepared using less expensive matrix material that is non-activeand generally regarded as safe. The net result is greatly improvedefficiency of drug delivery based on (1) reduced waste ofnon-deliverable active components incorporated into the non-needleportions of the microneedle array, and (2) higher drug concentration inthe skin penetrating needle tips. This technological advance results indramatically improved economic feasibility proportional to the cost ofdrug cargo, and increased effective cargo delivery capacity per needleof these novel microneedle arrays.

FIGS. 3A, 3B, 4A, and 4B illustrate various embodiments of microneedlearrays wherein the active component is concentrated in the microneedletips of the respective arrays. Thus, in contrast to conventionalmicroneedle arrays, the active component is not present at evenconcentration throughout the microneedle array since there is little orno active component present in the supporting base structure. Inaddition, in some embodiments (as shown, for example, in FIGS. 3A, 3B,4A, and 4B), not only is there little or no active component in thesupporting structures, the location of the active component isconcentrated in the upper half of the individual microneedles in thearray.

FIGS. 5A and 5B illustrate exemplary images of microneedles of amicroneedle array that contains active component concentrated in theupper half of the individual microneedles. The active component isillustrated as fluorescent particles that are concentrated in the tip ofthe microneedle, with the tip being defined by an area of themicroneedle that extends from a base portion in a narrowing and/ortapered manner. The base portion, in turn, extends from the supportingstructure of the array.

FIGS. 6A and 6B illustrate additional exemplary images of microneedlesof microneedle arrays that contain active components concentrated in theupper half of the individual microneedles. In FIG. 6A, the activecomponent, which is concentrated in the tip of the microneedles, isBSA-FITC. In FIG. 6B, the active component, which is also concentratedin the tip of the microneedles, is OVA-FITC.

As noted above, in some embodiments, individual microneedles cancomprise active components only in the upper half of the microneedle. Inother embodiments, individual microneedles can comprise activecomponents only in the tips or in a narrowing portion near the tip ofthe microneedle. In still other embodiments, individual needles cancomprise active components throughout the entire microneedle portionthat extends from the supporting structure.

The following embodiments describe various exemplary methods forfabricating microneedle arrays with one or more active componentconcentrated in the upper halves and/or tips of microneedles inrespective microneedle arrays.

Microneedle Arrays Fabricated by Sequential Micro-Molding andSpin-Drying Methods

The following steps describe an exemplary method of fabricatingmicroneedle arrays using sequential micro-molding and spin-drying.Active components/cargo can be prepared at a desired usefulconcentration in a compatible solvent. As described herein, the solventsof the active component(s) can be cargo specific and can comprise abroad range of liquids, including for example, water, organic polar,and/or apolar liquids. Examples of active components are discussed inmore detail below and various information about those active components,including tested and maximum loading capacity of various microneedlearrays are also discussed in more detail below.

If desired, multiple loading cycles can be performed to achieve higheractive cargo loads as necessary for specific applications. In addition,multiple active cargos can be loaded in a single loading cycle as acomplex solution, or as single solutions in multiple cycles (e.g.,repeating the loading cycle described below) as per specificcargo-compatibility requirements of individual cargos. Also, particulatecargos (including those with nano- and micro-sized geometries) can beprepared as suspensions at the desired particle number/volume density.

Example 1

a) As described in more detail below in the micromilling embodiments, anactive cargo's working stock solution/suspension can be applied to thesurface of microneedle array production molds at, for example, about 40μl per cm² surface area.

b) The microneedle array production molds with active cargo(s) can becentrifuged at 4500 rpm for 10 minutes to fill the microneedle arrayproduction molds needles with the working cargo stock.

c) The excess cargo solution/suspension can be removed and the surfaceof the microneedle array production molds, washed with 100 μl phosphatebuffer saline (PBS) per cm² mold-surface area, or with the solvent usedfor the preparation of the active cargo's working stock.

d) The microneedle array production molds containing the active cargostock solution/suspension in the needle's cavity can be spin-dried at3500 rpm for 30 minutes at the required temperature with continuespurging gas flow through the centrifuge at 0-50 L/min to facilitateconcentration of the drying active cargo(s) in the needle-tips. Thepurging gas can be introduced into the centrifuge chamber throughtubular inlets. Moisture content can be reduced using a dehumidifiertempered to the required temperature with recirculation into thecentrifuge chamber. The purging gas can be air, nitrogen, carbon dioxideor another inert or active gas as required for specific cargo(s). Theflow rate is measured by flow-meters and controlled by a circulatingpump device.

e) 100 μl 20% CMC90 hydrogel in H₂O can be added to the surfacemicroneedle array production molds' per cm² microneedle array productionmolds-area to load the structural component of the microneedle arraydevice.

f) The microneedle array production molds can be centrifuged at 4500 rpmfor 10 min at the required temperature without purging gas exchange inthe centrifuge chamber to fill up the microneedle array production moldsneedle cavities with the CMC90 hydrogel. This can be followed by a 30min incubation period to enable rehydration of the active cargo(s)previously deposited in the microneedle array tips.

g) The microneedle array production molds can centrifuged at 3500 rpmfor 3 hours or longer at the required temperature with 0-50 L/minconstant purging gas flow through the centrifuge chamber to spin-dry theMNA devices to less than 5% moisture content.

h) The dried microneedle array devices can then be separated from themicroneedle array production molds for storage under the desiredconditions. In some embodiments, CMC90 based devices can be storablebetween about 50° C. to −86° C.

Examples of fabricated tip-loaded active cargo carrying microneedlearrays can be seen in FIGS. 3A-6B.

Micromilled Master Molds and Spin-Molded Microneedle Arrays

In the following embodiments, micromilling steps are preformed to createmicroneedle arrays of various specifications. It should be understood,however, that the following embodiments describe certain details ofmicroneedle array fabrication that can be applicable to processes ofmicroneedle array fabrication that do not involve micromilling steps,including the process described above in the previous example.

In the following embodiments, apparatuses and methods are described forfabricating dissolvable microneedle arrays using master molds formed bymicromilling techniques. For example, microneedle arrays can befabricated based on a mastermold (positive) to production mold(negative) to array (positive) methodology. Micromilling technology canbe used to generate various micro-scale geometries on virtually any typeof material, including metal, polymer, and ceramic parts. Micromilledmastermolds of various shapes and configurations can be effectively usedto generate multiple identical female production molds. The femaleproduction molds can then be used to microcast various microneedlearrays.

FIG. 7 illustrates an example of a precision-micromilling system thatcan be used for fabricating a microneedle mastermold. Mechanicalmicromilling uses micro-scale (for example, as small as 10 μm) millingtools within precision computer controlled miniature machine-toolplatforms. The system can include a microscope to view the surface ofthe workpiece that is being cut by the micro-tool. The micro-tool can berotated at ultra-high speeds (200,000 rpm) to cut the workpiece tocreate the desired shapes. As noted above, the micromilling process canbe used to create complex geometric features with many kinds ofmaterial. Various types of tooling can be used in the micromillingprocess, including, for example, carbide micro-tools. In a preferredembodiment, however, diamond tools can be used to fabricate themicroneedle arrays on the master mold. Diamond tooling can be preferableover other types of tooling because it is harder than conventionalmaterials, such as carbide, and can provide cleaner cuts on the surfaceof the workpiece.

Mastermolds can be micromilled from various materials, including, forexample, Cirlex® (DuPont, Kapton® polyimide), which is the mastermoldmaterial described in the exemplary embodiment. Mastermolds can be usedto fabricate flexible production molds from a suitable material, such asSYLGARD® 184 (Dow Corning), which is the production material describedin the exemplary embodiment below. The mastermold is desirably formed ofa material that is capable of being reused so that a single mastermoldcan be repeatedly used to fabricate a large number of production molds.Similarly each production mold is desirably able to fabricate multiplemicroneedle arrays.

Mastermolds can be created relatively quickly using micromillingtechnology. For example, a mastermold that comprises a 10 mm×10 mm arraywith 100 microneedles can take less than a couple of hours and, in someembodiments, less than about 30 minutes to micromill Thus, a shortramp-up time enables rapid fabrication of different geometries, whichpermits the rapid development of microneedle arrays and also facilitatesthe experimentation and study of various microneedle parameters.

The mastermold material preferably is able to be cleanly separated fromthe production mold material and preferably is able to withstand anyheighted curing temperatures that may be necessary to cure theproduction mold material. For example, in an illustrated embodiment, thesilicone-based compound SYLGARD® 184 (Dow Corning) is the productionmold material and that material generally requires a curing temperatureof about 80-90 degrees Celsius.

Mastermolds can be created in various sizes. For example, in anexemplary embodiment, a mastermold was created on 1.8 mm thick Cirlex®(DuPont, Kapton® polyimide) and 5.0 mm thick acrylic sheets. Each sheetcan be flattened first by micromilling tools, and the location where themicroneedles are to be created can be raised from the rest of thesurface. Micro-tools can be used in conjunction with a numericallycontrolled micromilling machine (FIG. 1) to create the microneedlefeatures (e.g., as defined by the mastermold). In that manner, themicromilling process can provide full control of the dimensions,sharpness, and spatial distribution of the microneedles.

FIG. 8 is an image from a scanning electron microscope (SEM) showing thestructure of a micromilled mastermold with a plurality of pyramidalneedles. As shown in FIG. 8, a circular groove can be formed around themicroneedle array of the mastermold to produce an annular (for example,circular) wall section in the production mold. The circular wall sectionof the production mold can facilitate the spincasting processesdiscussed below. Although the wall sections illustrated in FIG. 9 andthe respective mastermold structure shown in FIG. 8 is circular, itshould be understood that wall sections or containment means of othergeometries can be provided. For example, depending on what shape isdesired for the microneedle array device, the containment means can beformed in a variety of shapes including, for example, square,rectangular, trapezoidal, polygonal, or various irregular shapes.

As discussed above, the production molds can be made from SYLGARD® 184(Dow Corning), which is a two component clear curable silicone elastomerthat can be mixed at a 10:1 SYLGARD® to curing agent ratio. The mixturecan be degassed for about 10 minutes and poured over the mastermold toform an approximately 8 mm layer, subsequently degassed again for about30 minutes and cured at 85° C. for 45 minutes. After cooling down toroom temperature, the mastermold can be separated from the curedsilicone, and the silicone production mold trimmed to the edge of thecircular wall section that surrounds the array (FIG. 9). From a singlemastermold, a large number of production molds (e.g., 100 or more) canbe produced with very little, if any, apparent deterioration of theCirlex® or acrylic mastermolds.

FIG. 9 is an SEM image of a pyramidal production mold created asdescribed above. FIG. 10 illustrates an enlarged segment of theproduction mold with a pyramidal needle molding well in the center ofthe image. The molding well is configured to receive a base material(and any components added to the base material) to form microneedleswith an external shape defined by the molding well.

To construct the microneedle arrays, a base material can be used to formportions of each microneedle that have bioactive components and portionsthat do not. As discussed above, each microneedle can comprise bioactivecomponents only in the microneedles, or in some embodiments, only in theupper half of the microneedles, or in other embodiments, only in aportion of the microneedle that tapers near the tip. Thus, to controlthe delivery of the bioactive component(s) and to control the cost ofthe microneedle arrays, each microneedle preferably has a portion with abioactive component and a portion without a bioactive component. In theembodiments described herein, the portion without the bioactivecomponent includes the supporting structure of the microneedle arrayand, in some embodiments, a base portion (e.g., a lower half) of eachmicroneedle in the array.

Various materials can be used as the base material for the microneedlearrays. The structural substrates of biodegradable solid microneedlesmost commonly include poly(lactic-co-glycolic acid) (PLGA) orcarboxymethylcellulose (CMC) based formulations; however, other basescan be used.

CMC is generally preferable to PLGA as the base material of themicroneedle arrays described herein. The PLGA based devices can limitdrug delivery and vaccine applications due to the relatively hightemperature (e.g., 135 degrees Celsius or higher) and vacuum requiredfor fabrication. In contrast, a CMC-based matrix can be formed at roomtemperature in a simple spin-casting and drying process, makingCMC-microneedle arrays more desirable for incorporation of sensitivebiologics, peptides, proteins, nucleic acids, and other variousbioactive components.

CMC-hydrogel can be prepared from low viscosity sodium salt of CMC withor without active components (as described below) in sterile dH₂O. Inthe exemplary embodiment, CMC can be mixed with sterile distilled water(dH₂O) and with the active components to achieve about 25 wt % CMCconcentration. The resulting mixture can be stirred to homogeneity andequilibrated at about 4 degrees Celsius for 24 hours. During thisperiod, the CMC and any other components can be hydrated and a hydrogelcan be formed. The hydrogel can be degassed in a vacuum for about anhour and centrifuged at about 20,000 g for an hour to remove residualmicro-sized air bubbles that might interfere with a spincasting/dryingprocess of the CMC-microneedle arrays. The dry matter content of thehydrogel can be tested by drying a fraction (10 g) of it at 85 degreesCelsius for about 72 hours. The ready-to-use CMC-hydrogel is desirablystored at about 4 degrees Celsius until use.

Active components can be incorporated in a hydrogel of CMC at arelatively high (20-30%) CMC-dry biologics weight ratio before thespin-casting process. Arrays can be spin-cast at room temperature,making the process compatible with the functional stability of astructurally broad range of bioactive components. Since the master andproduction molds can be reusable for a large number of fabricationcycles, the fabrication costs can be greatly reduced. The resultingdehydrated CMC-microneedle arrays are generally stable at roomtemperature or slightly lower temperatures (such as about 4 degreesCelsius), and preserve the activity of the incorporated biologics,facilitating easy, low cost storage and distribution.

In an exemplary embodiment, the surface of the production molds can becovered with about 50 μl (for molds with 11 mm diameter) of CMC-hydrogeland spin-casted by centrifugation at 2,500 g for about 5 minutes. Afterthe initial CMC-hydrogel layer, another 50 μl CMC-hydrogel can belayered over the mold and centrifuged for about 4 hours at 2,500 g. Atthe end of a drying process, the CMC-microneedle arrays can be separatedfrom the molds, trimmed off from excess material at the edges, collectedand stored at about 4 degrees Celsius. The production molds can becleaned and reused for further casting of microneedle arrays.

In some embodiments, CMC-solids can be formed with layers that do notcontain active components and layers that contain active components.FIGS. 11A-D illustrate CMC-solids with different shapes (FIGS. 11A and11B) and embedded active cargos on an upper layer which becomes, aftermicromilling, the portions of the microneedle with the activecomponents. FIG. 11C illustrates micron sized fluorescent particleslayered on a surface of a non-active component containing layer and FIG.11D illustrates toluidine blue examples layered on a surface of anon-active component containing layer.

FIGS. 12A and 12B also illustrate CMC-solids with different shapes, withFIG. 12B showing a square shape and FIG. 12B showing a rectangularshape. Both CMC solids can be milled to dimensions for furtherprocessing as described herein. It should be understood that thegeometries and the active cargo shown herein are not intended to belimited to the exemplary embodiments.

Example 2

CMC-solids can be prepared with defined geometry and active cargocontents in one or more layers of the prepared structure. Examples ofactive cargos integrated into CMC-solids are described more detailherein. Upon construction of the CMC-solids with embedded active cargocontained in at least one layer of the CMC-solid, the CMC solids can bemilled to project-specific dimensions and micro-milled to fabricatemicroneedle devices as described herein.

Example 3

In another embodiment, one or more layers of active cargo can beembedded on CMC-solids for direct micromilling of the microneedle array.FIG. 13 illustrates a sample representation of vertical multi-layereddeposition and CMC embedding of active cargos on CMC-solids for directmicro-milling of MNA devices.

In one exemplary method, microneedle arrays can be fabricated bypreparing CMC-solids with a defined geometries and without any activecargo contained therein. Then, blank CMC-solids can be milled to adesired dimension.

As shown in FIG. 13, active cargo(s) can be deposited onto the CMC-solidin project specific geometric patterns for inclusion of the activecargo(s) specifically in the tips of micro-milled MNA devices.

The methods active cargo deposition onto the CMC-solid blank caninclude, for example:

1) Direct printing with micro-nozzle aided droplet deposition.

2) Transfer from preprinted matrices.

3) Droplet-deposition with computer controlled robotic systems.

FIG. 14 illustrates layering and spatial distribution of embedded activecargos in a CMC-solid block. After the first layer is deposited (A) itcan be covered with a CMC layer (B) that provides the surface for thesubsequent deposition of the active cargo (C). The process can berepeated until all desired layers are deposited and encased in a solidCMC-block suitable for the micro-milling process (D-F).

FIG. 15 illustrates a schematic view of a cross-section of a CMC-blockencasing the deposits of the active cargo in a spatially controlledmanner (A). The method allows 3-dimensional control and placement of theactive components after micro-milling in the MNA-device (B). In panel(B) of FIG. 15, the placement of the active cargos are shown in thestems of the active cargo; however through the control of the millingprocess the placement can be controlled vertically from the tip to thebase of the microneedles. Colors represent different active componentsor different amount/concentration of the same material.

Thus, a method of vertically layered deposition of active cargos inmicroneedles is provided by depositing one or more active cargossequentially on the surface of the CMC-solids in contact with each otheror separated by layers of CMC. In some embodiments, horizontal patterndeposition of the active cargos can result in spatial separation of thecargos. By combining vertical and horizontal patterning of active cargodeposition, 3 dimensional delivery and distribution of each of thedefined active components can be achieved, further reducing waste ofactive components during fabrication of microneedle arrays.

Microneedle Integrated Adenovectors

The following embodiments are directed to dissolvable microneedlearrays, such as those described herein, that incorporate infectiousviral vectors into the dissolvable matrix of microneedle arrays. Usingthis technology, for the first time, living viral vectors can beincorporated into microneedle arrays. As described herein, theincorporation of viral vectors within the disclosed microneedle arraysstabilizes the viral vectors so that they maintain their infectivityafter incorporation and after prolonged periods of storage. Theapplication of microneedle array incorporated adenovectors (MIAs) to theskin results in transfection of skin cells. In a vaccine setting, wehave demonstrated that skin application of MIAs encoding an HIV antigenresults in potent HIV specific immune responses. These results aredescribed in detail in the examples below.

Example 4

The microneedle integrated adenovectors preparation method describedherein preserves the viability of the adenoviral particles during thepreparation and in dry storage. These steps were specifically designedbased on the physical and chemical properties of CMC microneedle arrays.Viral viability in CMC microneedle arrays was achieved by

-   -   Inclusion of low viscosity carboxymethyl cellulose (CMC90) at        2.5% final concentration (step 2.) and by    -   Timed and temperature controlled spin-drying concentration of        the adenoviral particles in the tips of the microneedle array        devices (step 6).    -   Controlled partial rehydration of the needle-tip loaded        adenoviral particles (step 8.)

Preparation of Tip-loaded Microneedle Integrated Adenovectors (MIAs):

1) Resuspend adenoviral particles at 2×109 particles/ml density inTrehalose-storage buffer (5% trehalose Sigma-Aldrich USA, 20 mM TrispH7.8, 75 mM NaCl, 2 mM MgCl2, 0.025% Tween 80)

2) Mix resuspended viral stock with equal volume of 5% CMC90 prepared inTrehasole-storage buffer, resulting in a 1×109 particles/ml densityadenoviral working stock.

3) Add adenoviral working stock suspension to the surface of microneedlearray production molds (as described in detail in other embodimentsherein) at 40 μl per cm2 surface area.

4) The molds are centrifuged at 4500 rpm for 10 minutes at 22° C. tofill the needle tips with adenoviral working stock.

5) The excess viral stock is removed and the surface of the molds washedwith 100 μl (phosphate buffer saline (PBS) solution per cm2 mold-surfacearea.

6) The microneedle array-molds containing the adenoviral stock solutiononly in the needle's cavity are partially spin-dried at 3500 rpm for 10minutes at 22° C.

7) 100 μl 20% structural, non-cargo containing CMC90 hydrogel in H₂Oadded to the surface microneedle array-molds' per cm2 mold-area to formthe structure of the MIA device.

8) Centrifuge at 4500 rpm for 10 min at 22° C. to fill up the needlecavities with 20% CMC90 and allow 30 min incubation for the rehydrationof the adenoviral particles dried in the tips (step 3-6, above).

9) By centrifugation spin-dry the MIA devices to less than 5% moisturecontent at 3500 rpm for 3 hours at 22° C. with 10 L/min constant airflow through the centrifuge chamber.

10) De-mold the dried MIA devices for storage at 4° C. or −80° C.

Example 5

We have evaluated the potency and stability MNA incorporated recombinantadenoviral particles. Ad5.EGFP was incorporated into CMC hydrogel MNAsto fabricate a final product that contained 1010 virus particles/MNA.Control blank MNAs were prepared identically but without the virus.Batches of Ad5.EGFP and control MNAs were stored at RT, 4° C. and at−86° C. and viral stability was evaluated in infectious assays. Specifictransduction activity of the MNA incorporated Ad5.EGFP virus wasassessed in vitro using 293T cells. Cells were plated at 2×106/well insix well plates and transduced in duplicate with diluted virussuspension, suspension+empty MNA (control), or Ad5.EGFP MNAs stored atRT, 4° C. and −86° C. for the indicated time periods. As a negativecontrol untransduced wells were included. Initially cell populationswere analyzed after 24 h by flow cytometry for GFP expression(representative histogram is shown in FIG. 35).

As shown in FIG. 35, the incorporation of Ad5.EGFP into MNAs does notreduce transduction efficiency. Flow cytometry analysis of GFPexpressing target 293T cells 24 h after transduction with identicaltiters of Ad5.EGFP either in suspension or incorporated into CMC-patchesvs. untransfected control cells. FIG. 36 shows the stability of MNAembedded Ad5.EGFP virus. GFP gene expression was assayed by flowcytometry as in FIG. 37 and normalized to the infection efficiency of−86° C. preserved Ad5.EGFP suspension.

It has been found that the infection efficiency using MNA Ad5.EGFP viruswas 87.92±4.5%, which is similar to that observed for traditional −86°C. preserved Ad5.EGFP suspension (FIGS. 35 and 36), suggesting that themanufacturing process does not adversely affect the transductionefficiency of Ad-EGFP viral particles. To asses infectivity over time,the transfection efficiency of freshly prepared −86° C. preservedAd5.EGFP suspensions was compared to that of MNA incorporated Ad5.EGFPstored for prolonged periods of time at either RT, 4 C, or −86 C.Infectivity (normalized to Ad5.EGFP suspension+empty CMC-patch) isreported for storage periods of up to 365 days (FIG. 36). These resultssuggest that the infectiousness of MNA Ad5.EGFP is remarkably stablewith storage at either 4 C or −86 C, and somewhat stable at RT for up to30 days.

These results demonstrate that microneedle array delivered Ad transgenesare expressed in the skin and induce potent cellular immune responses.To specifically evaluate gene expression in vivo, we determined GFPexpression in skin following either traditional intradermal injection(I.D.) or microneedle array-mediated intracutaneous delivery. Wedelivered 108 Ad5.GFP viral particles by ID injection or topically via asingle microneedle array application (FIG. 37). Skin was harvested 48 hlater, cryosectioned, counter-stained using blue fluorescent DAPI toidentify cell nuclei, and then imaged by fluorescent microscopy.Significant cellular GFP expression was observed following both I.D. andmicroneedle array delivery. To evaluate immunogenicity, we evaluatedantigen-specific lytic activity in vivo following a single I.D. ormicroneedle array immunization without boosting. For this purpose weimmunized groups of mice with E1/E3-deleted Ad5-based vectors thatencode codon-optimized SIVmac239 gag full-length or SIVmac239 gag p17antigens (Ad5.SIV gag, Ad5.SIV gag p17). Empty vector was used as acontrol (Ad5). We observed potent and similar levels of in vivo lyticactivity specific for the dominant SIVgag p17-derived peptide KSLYNTVCV(SIVmac239 gag 76-84) following either I.D. or microneedle arrayimmunization with either Ad5.SIV gag or Ad5.SIV gag p17 (FIG. 37, CTL).

The microneedle array technology disclosed herein can also facilitateclinical gene therapy. It addresses, for example, at least two majorlimitations of conventional approaches. First, it enables stabilizationand storage of recombinant viral vectors for prolonged periods of time.By rendering live virus vectors resistant to high and low temperatureswith proven seroequivalence to frozen liquid formulations, microneedlearray stabilization will relieve pressures related to the ‘cold chain.’Further, integration in microneedle arrays enables precise, consistentand reproducible dosing of viral vectors not achievable by conventionalmethods. Finally, the viral vector is repackaged in the only necessarydelivery device, the biocompatible and completely disposable microneedlearray that directs delivery precisely to the superficial layers of theskin. Such a gene delivery platform is useful in providingpatient-friendly, clinical gene therapy.

Since these microneedle arrays have been engineered to not penetrate tothe depth of vascular or neural structures, gene delivery to human skinwill be both painless and bloodless. In addition, the fabricationprocess is flexible, enabling simple and rapid low cost production withefficient scale-up potential. Also, as a final product, the MIA deviceit is stable at room temperature and is inexpensive to transport andstore. In combination, these structural and manufacturing advantages canenable broad and rapid clinical deployment, making this gene deliverytechnology readily applicable to the prevention and/or treatment of abroad range of human diseases. Moreover, this approach can be extendedto other vector-based vaccine platforms that are currently restricted bythe same limitations (e.g., vaccinia virus, AAV etc.). For at leastthese reasons, the disclosed microneedle arrays and methods of using thesame significantly advance the recombinant gene therapy field.

Microneedle Arrays—Exemplary Active Components

Various active components are described in detail below. Forconvenience, the following examples are based on a microneedle arraywhich is 6.3×6.3 mm. This size, and hence cargo delivery can be variedby increasing or decreasing 2-100 fold.

General considerations for the maximum active cargo quantities include,for example, total needle volume in the array and solubility of theactive component(s) in the solvent (generally expected to be <50%).

Tip Loaded Amount Tip Loaded Max. predicted Components: into MNA deviceloading capacity μg/device (unless indicated differently)

Live viruses⁽¹⁾ Ad5.GFP 5 × 10⁸ 2-5 × 10⁹ (adeno viral particles/MNAparticles/MNA GFP expression vector) Ad-SIVgag 5 × 10⁸ 2-5 × 10⁹ (adenoviral particles/MNA particles/MNA gag expression vector) Ad-SIVp17 5 ×10⁸ 2-5 × 10⁹ (adeno viral particles/MNA particles/MNA gag-p17expression vector) Ψ5 5 × 10⁸ 2-5 × 10⁹ (non-recombinant Adparticles/MNA particles/MNA vector) Lenti-GFP⁽²⁾ 5 × 10⁶ 2-5 × 10⁷(Lenti viral GFP particles/MNA particles/MNA expression vector)Vaccinia virus (immunization)Recombinant vaccinia virus (gene therapy, genetic engineering)Seasonal influenzaMMR (Measles, Mumps, Rubella)

Proteins/Peptides BSA (FITC labeled) 240 400 OVA (FITC labeled) 100 400OVA (no label) 240 400 Flu (split vaccine) 0.22 (2-5)

Epitope Peptides⁽³⁾ TRP-2 50 200 EphA2 (a) 50 400 EphA2 (b) 50 400 DLK-150 200 Multiple epitopes 200 400-600 in one MNA Substance-P 15 (NK-1Rligand)

Nucleic acids CpG 1668 120 250 CpG 2006 120 250 Poly(I:C) 250 250Plasmid vectors 100 200 (High mol. weight DNA)

Peptides/Nucleic acid combos OVA/CpG 250/120 OVA/CpG/poly(I:C)250/120/250 Epitope 200/250 peptides/poly(I:C)

Organics Doxorubicin 1 to 1000 ug R848 (TLR7/8 ligand) 6 L733 2 (NK-1antagonist) DNCB (irritant) 100

Particulates Micro-particles 1 × 10⁶ 2-5 × 10⁷ (1μ diameterparticles/MNA particles/MNA microsphares)Nano Scale ParticlesPLG/PLA Based

Other Biologic tumor lysate/CpG 250/120 tumor lysate/CpG/poly(I:C)256/120/250 tumor lysates/poly(I:C) 200/250Tip-loading of live adenoviruses generally includes the followingmodifications:

a) The presence of 5% trehalose and 2.5% CMC90 in the tip-loadinghydrogel suspension.

b) The temperature of the process is maintained at 22° C.

In addition, Lenti viral vectors generally require 4° C. processing andvapor trap based humidity controls. Also, short epitope peptidesgenerally are solubilized in DMSO, with the evaporation time of thesolvent during tip-loading is 4 hours.

Microneedle Structures and Shapes

For each of the embodiments below, it should be understood that one ormore layers of active components can be provided in the microneedles ofthe microneedle arrays as described above. Thus, for example, in someembodiments, active components are only provided in the area of themicroneedle—not in the structural support of the array, such as shown inFIG. 15. Moreover, in other embodiments, the active components areconcentrated in the upper half of the microneedles, such as in the tipsof the microneedles as shown in FIGS. 3A-4B.

FIGS. 16A and 16B are SEM images of a CMC-microneedle array formed witha plurality of pyramidal projections (i.e., microneedles). The averagetip diameter of the pyramidal needles shown in FIG. 16A is about 5-10μm. As shown in FIG. 16B, the sides of the pyramidal needles can beformed with curved and/or arcuate faces that can facilitate insertion inskin.

FIG. 17 is another SEM image of a single needle of a microneedle array.The microneedle shown in FIG. 17 is a base-extended pillar type moldedCMC-microneedle. The base-extended pillar type microneedle comprises abase portion, which is generally polyagonal (for example, rectangular)in cross section, and a projecting portion that extends from the baseportion. The projecting portion has a lower portion that issubstantially rectangular and tip portion that generally tapers to apoint. The tip portion is generally pyramidal in shape, and the exposedfaces of the pyramid can be either flat or arcuate. The projectingportion can be half or more the entire length of the needle.

FIGS. 18 and 19 illustrate micrographs of pyramidal (FIG. 18) and pillartype (FIG. 19) molded CMC-microneedles. Because the pyramidal needleshave a continually increasing cross-sectional profile (dimension) fromthe needle point to the needle base, as the needle enters the skin, theforce required to continue pushing the pyramidal needle into the skinincreases. In contrast, pillar type needles have a generally continuouscross-sectional profile (dimension) once the generally rectangularportion of the projection portion is reached. Thus, pillar type needlescan be preferable over pyramidal type needles because they can allow forthe introduction of the needle into the skin with less force.

FIG. 20 illustrates schematic representation of microneedle shapes andstructures that are generally suitable for fabrication by spin-castingmaterial into a mastermold formed by micromilling Since the shapes andstructures shown in FIG. 20 do not contain any undercuts, they generallywill not interfere with the molding/de-molding process. The structuresin FIG. 20 include (a) a generally pyramidal microneedle, (b) a “sharp”pillar type microneedle (without the base member of FIG. 8), (c) a“wide” pillar type microneedle, (d) a “short” pillar type microneedle(having a short pillar section and a longer pointed section), and (e) a“filleted” pillar type microneedle.

While the volume of the pyramidal microneedles can be greater than thatof the pillar type microneedles, their increasing cross-sectionalprofile (dimension) requires an increasing insertion force. Accordingly,the geometry of the pyramidal microneedles can result in reducedinsertion depths and a reduced effective delivery volume. On the otherhand, the smaller cross-sectional area and larger aspect ratio of thepillar microneedles may cause the failure force limit to be lower. Thesmaller the apex angle α, the “sharper” the tip of the microneedle.However, by making the apex angle too small (e.g., below about 30degrees), the resulting microneedle volume and mechanical strength maybe reduced to an undesirable level.

The penetration force of a microneedle is inversely proportional to themicroneedle sharpness, which is characterized not only by the included(apex) angle of the microneedles, but also by the radius of themicroneedle tip. While the apex angle is prescribed by the mastermoldgeometry, the tip sharpness also depends on the reliability of the mold.Micromilling of mastermolds as described herein allows for increasedaccuracy in mold geometry which, in turn, results in an increasedaccuracy and reliability in the resulting production mold and themicroneedle array formed by the production mold.

The increased accuracy of micromilling permits more accurate anddetailed elements to be included in the mold design. For example, asdiscussed in the next section below, the formation of a fillet at thebase of a pillar type microneedle can significantly increase thestructural integrity of the microneedle, which reduces the likelihoodthat the microneedle will fail or break when it impacts the skin. Whilethese fillets can significantly increase the strength of themicroneedles, they do not interfere with the functional requirements ofthe microneedles (e.g., penetration depth and biologics volume). Suchfillets are very small features that can be difficult to create in amaster mold formed by conventional techniques. However, the micromillingtechniques described above permit the inclusion of such small featureswith little or no difficulty.

Mechanical Integrity and Penetration Capabilities

Microneedle arrays are preferably configured to penetrate the stratumcorneum to deliver their cargo (e.g., biologics or bioactive components)to the epidermis and/or dermis, while minimizing pain and bleeding bypreventing penetration to deeper layers that may contain nerve endingsand vessels. To assess the mechanical viability of the fabricatedmicroneedle arrays, tests were performed on the pyramidal and pillartype microneedle arrays as representative variants of array geometry(shown, e.g., in FIGS. 7B and 8). The first set of tests illustrate thefailure limit of microneedles, and include pressing the microneedlearray against a solid acrylic surface with a constant approach speed,while simultaneously measuring the force and the displacement untilfailure occurs. The second set of tests illustrate the piercingcapability of the microneedles on human skin explants.

FIG. 21 illustrates a test apparatus designed for functional testing.The sample (i.e., microneedle array) was attached to a fixture, whichwas advanced toward a stationary acrylic artifact (PMMA surface) at aconstant speed of about 10 mm/s speed using a computer-controlled motionstage (ES14283-52 Aerotech, Inc.). A tri-axial dynamometer (9256C1,Kistler, Inc.) that hosted the acrylic artifact enabled high-sensitivitymeasurement of the forces.

FIG. 22 illustrates force-displacement curves of data measured duringfailure tests. The curve on the left is representative of data obtainedfrom testing a pillar microneedle sample and the curve on the right isrepresentative of data obtained from testing a pyramid microneedle. Asseen in FIG. 22, the failure of these two kinds of microneedles aresignificantly different; while the pyramidal arrays plastically deform(bend), the pillar type arrays exhibit breakage of the pillars at theirbase. This different failure behavior lends itself to considerablydifferent displacement-force data. The failure (breakage) event can beeasily identified from the displacement-force data as indicated in thefigure. Based on the obtained data, the failure point of pillar typemicroneedles was seen to be 100 mN in average. As only about 40 mN offorce is required for penetration through the stratum corneum, themicroneedles are strong enough to penetrate human skin without failure.Furthermore, since parallelism between microneedle tips and the acrylicartifact cannot be established perfectly, the actual failure limit willlikely be significantly higher than 100 mN (i.e., microneedles broke ina successive manner, rather than simultaneous breakage of most/allmicroneedles).

The pyramidal microneedles presented a continuously increasing forcesignature with no clear indication of point of failure. To identify thefailure limit for the pyramidal microneedles, interrupted tests wereconducted in which the microneedles were advanced into the artifact by acertain amount, and retreated and examined through optical microscopeimages. This process was continued until failure was observed. For thispurpose, the failure was defined as the bending of the pyramidalmicroneedles beyond 15 degrees.

To further analyze the failure of the microneedles, the finite-elementsmodel (FEM) of the microneedle arrays shown in FIG. 23 was developed. Toobtain the mechanical properties (elastic modulus and strength limit) ofthe CMC material, a series of nanoindentation tests (using a Hysitronnanoindentor). The average elastic modulus and yield strength of the CMCmaterial (as prepared) were 10.8 GPa and 173 MPa, respectively. Thisindicates that the prepared CMC material has a higher elastic modulusand yield strength than both PMMA (elastic modulus: 3.1 GPa, yieldstrength: 103 MPa) and polycarbonate (elastic modulus: 2.2 GPa, yieldstrength: 75 MPa), indicating the superior strength and stiffness of CMCmaterial with respect to other polymers.

Using this data, a series of FEM simulations were conducted. It waspredicted from the 1-BM models that failure limit of pyramidal andsharp-pillar (width=134 μm) microneedles with 600 μm height, 30 degreeapex angle, and 20 μm fillet radius were 400 mN (pyramid) and 290 mN(sharp-pillar) for asymmetric loading (5 degrees loadingmisorientation). Considering that the minimum piercing force requirementis about 40 mN, pyramid and sharp-pillar microneedles would have factorsof safety of about 10 and 7.25, respectively.

When the fillet radius is doubled to 40 μm, the failure load for thepillar was increased to 350 mN, and when the fillet radius is reduced to5 μm, the failure load was reduced to 160 mN, which is close to theexperimentally determined failure load. The height and width of thepillars had a significant effect on failure load. For instance, for 100μm width pillars, increasing the height from 500 μm to 1000 μm reducedthe failure load from 230 mN to 150 mN. When the width is reduced to 75μm, for a 750 μm high pillar, the failure load was seen to be 87 mN.

To evaluate penetration capability, pyramidal and sharp-pillarmicroneedle arrays were tested for piercing on water-based model elasticsubstrates and on full thickness human skin. FIG. 24 illustrates stereomicrographs of pyramidal (Panels A, C, and E) and pillar typemicroneedle arrays (B, D, and F) after 4 minutes of exposure to modelelastics. In particular, toluene blue tracer dye was deposited in modelelastic substrates (Panels C and D) or freshly excised full thicknesshuman skin explants (Panels E and F) after application of pyramidal orpillar type microneedle arrays.

The model elastic substrate comprised about 10% CMC and about 10%porcine gelatin in PBS gelled at about 4 degrees Celsius for about 24hours or longer. The surface of the elastics was covered with about 100μm thick parafilm to prevent the immediate contact of the needle-tipsand the patch materials with the water based model elastics. To enablestereo microscopic-imaging, trypan blue tracer dye (Sigma Chem., cat #T6146) was incorporated into the CMC-hydrogel at 0.1% concentration. Thepatches were applied using a spring-loaded applicator and analyzed afterabout a 4 minute exposure. Based on physical observation of the dye inthe target substrates, the dissolution of the microneedles of the twodifferent geometries was markedly different.

The sharp-pillar needles applied to the model elastic substrate releasedsubstantially more tracer dye to the gel matrix than that observed forthe pyramidal design (FIG. 24, C vs. D). Images of the recovered patches(FIG. 24, A vs. B) were consistent with this observation, as thedegradation of the sharp-pillar needles was more advanced than that ofthe pyramidal needles. To extrapolate this analysis to a more clinicallyrelevant model, pyramidal and pillar type microneedle arrays wereapplied to freshly excised full thickness human skin explants using thesame force from the spring loaded applicator. Consistent with resultsfrom the elastic model, the pyramidal microneedle arrays depositedvisibly less tracer dye than the sharp-pillar microneedle arrays (FIG.24, E vs. F).

To further evaluate penetration and to assess delivery effectiveness tohuman skin, CMC-microneedle arrays were fabricated with BioMag(Polysciences, Inc., cat#. 84100) beads or fluorescent particulatetracers (Fluoresbrite YG 1 μm, Polysciences Inc., cat#. 15702). Thepyramidal CMC-microneedle arrays containing fluorescent or solidparticulates were applied to living human skin explants as describedpreviously. Five minutes after the application, surface residues wereremoved and skin samples were cryo-sectioned and then counterstainedwith toluene blue for imaging by light microscopy (FIGS. 25A and 25B) orby fluorescent microscopy (FIG. 25C).

Pyramidal CMC-microneedles effectively penetrated the stratum corneum,epidermis, and dermis of living human skin explants, as evidenced by thedeposition of Biomag beads lining penetration cavities corresponding toindividual needle insertion points (representative sections shown inFIGS. 25A and 25B). In particular, ordered cavities (FIG. 25A, cavitiesnumbered 1-4, toluene blue counterstain, 10×) and deposits of BioMagparticles (brown) lining penetration cavities were evident (FIG. 25B,40×), indicating microneedle penetrated of human skin. Further, analysisof sections from living human explants stained with DAPI to identifycell nuclei and anti-HLA-DR to identify MHC class II+ antigen presentingcells revealed high density fluorescent particulates deposited in thesuperficial epidermis and dermis, including several particlesco-localized with class II+ antigen presenting cells (FIG. 25C, DAPI(blue), HLA-DR+(red) and fluorescent particles (green), 40×).

These results further demonstrate that the CMC microneedle arraysdescribed herein can effectively penetrate human skin and deliverintegral cargo (bioactive components), including insoluble particulates.They are consistent with effective delivery of particulate antigens toantigen presenting cells in human skin, currently a major goal ofrational vaccine design.

To further address microneedle array delivery in vivo, the cutaneousdelivery of particulate antigen in vivo was modeled by similarlyapplying fluorescent particle containing arrays to the dorsal aspect ofthe ears of anesthetized mice. After 5 minutes, patches were removed andmice resumed their normal activity. Three hours or 3 days, ear skin anddraining lymph nodes were analyzed for the presence of fluorescentparticles. Consistent with observations of human skin, particulates wereevident in the skin excised from the array application site (data notshown). Further, at the 3 day time point, substantial numbers ofparticles were evident in the draining lymph nodes. FIGS. 26A and 26Billustrates substantial numbers of particles that were evident in thedraining lymph Nodes (FIG. 26A, 10×), including clusters of particulatesclosely associated with Class II+ cells (FIG. 26B, 60×) suggesting thepresence of lymph node resident antigen presenting cells withinternalized particulates.

To quantitatively evaluate the effects of needle geometry on cargodelivery using microneedle arrays, 3H-tracer labeled CMC-microneedlearrays were constructed. The CMC-hydrogel was prepared with 5% wtovalbumin as a model active component at 25 wt % final dry weightcontent (5 g/95 g OVA/CMC) and trace labeled with 0.1 wt % trypan blueand 0.5×106 dpm/mg dry weight 3H-tracer in the form of 3H-thymidine (ICNInc., cat #2406005). From a single batch of labeledCMC-hydrogel-preparation four batches of 3H-CMC-microneedle arrays werefabricated, containing several individual patches of pyramidal andsharp-pillar needle geometry. The patches were applied to human skinexplants as described above and removed after 30 min exposure. Thepatch-treated area was tape-striped to remove surface debris and cutusing a 10 mm biopsy punch. The 3H content of the excised human skinexplants-discs was determined by scintillation counting. The specificactivity of the 3H-CMC-microneedle patch-material was determined andcalculated to be 72,372 cpm/mg dry weight. This specific activity wasused to indirectly determine the amount of ovalbumin delivered to andretained in the skin. The resulting data is summarized in Table 1 below.

The tested types of patches were consistent from microneedle array tomicroneedle array (average standard deviation 24-35%) and batch to batch(average standard deviation 7-19%). The intra-batch variability for bothneedle geometry was lower than the in-batch value indicating that theinsertion process and the characteristics of the target likely plays aprimary role in the successful transdermal material delivery andretention. The patch-material retention data clearly demonstrate theforemost importance of the microneedle geometry in transdermal cargodelivery. Pillar-type needle geometry afforded an overall 3.89 foldgreater deposition of the 3H labeled needle material than that of thepyramidal needles. On the basis of the deposited radioactive material,it is estimated that the pyramidal needles were inserted about 200 μmdeep while the pillar-type were inserted about 400 μm or more.

TABLE 4.2.5. Transfer of ³H-labeled CMC-microneedle material into humanskin explants by pyramidal and pillar-type needles. Pyramid PyramidalNeedles Pillar-Type Pillar-Type Needles Pillar to Array Needles STDevOVA Transferred Needles STDev OVA Transferred Pyramid Batches(cpm/patch) (%) (μg/patch) (cpm/patch) (%) (μg/patch) Ratio Batch A2459.00 17.56 1.70 11700.50 31.52 8.08 4.76 Batch B 3273.50 57.39 2.2612816.50 21.45 8.85 3.92 Batch C 2757.75 46.13 1.90 12240.00 26.77 8.464.44 Batch D 3782.00 36.27 2.61 10921.50 9.32 7.55 2.89 IntraBatch3068.06 19.00 2.12 11919.63 6.77 8.24 3.89 AVG

Desirably, the microneedle arrays described herein can be used forcutaneous immunization. The development of strategies for effectivedelivery of antigens and adjuvants is a major goal of vaccine design,and immunization strategies targeting cutaneous dendritic cells havevarious advantages over traditional vaccines.

The microneedle arrays described herein can also be effective inchemotherapy and immunochemotherapy applications. Effective and specificdelivery of chemotherapeutic agents to tumors, including skin tumors isa major goal of modern tumor therapy. However, systemic delivery ofchemotherapeutic agents is limited by multiple well-establishedtoxicities. In the case of cutaneous tumors, including skin derivedtumors (such as basal cell, squamous cell, Merkel cell, and melanomas)and tumors metastatic to skin (such as breast cancer, melanoma), topicaldelivery can be effective. Current methods of topical delivery generallyrequire the application of creams or repeated local injections. Theeffectiveness of these approaches is currently limited by limitedpenetration of active agents into the skin, non-specificity, andunwanted side effects.

The microneedle arrays of the present disclosure can be used as analternative to or in addition to traditional topical chemotherapyapproaches. The microneedle arrays of the present disclosure canpenetrate the outer layers of the skin and effectively deliver theactive biologic to living cells in the dermis and epidermis. Delivery ofa chemotherapeutic agents results in the apoptosis and death of skincells.

Further, multiple bioactive agents can be delivered in a singlemicroneedle array (patch). This enables an immunochemotherapeuticapproach based on the co-delivery of a cytotoxic agent with and immunestimulant (adjuvants). In an immunogenic environment created by theadjuvant, tumor antigens releases from dying tumor cells will bepresented to the immune system, inducing a local and systemic anti-tumorimmune response capable of rejecting tumor cells at the site of thetreatment and throughout the body.

In an exemplary embodiment, the delivery of a biologically active smallmolecule was studied. In particular, the activity of thechemotherapeutic agent Cytoxan® delivered to the skin with CMCmicroneedle arrays was studied. The use of Cytoxan® enables directmeasurement of biologic activity (Cytoxan® induced apoptosis in theskin) with a representative of a class of agents with potential clinicalutility for the localized treatment of a range of cutaneousmalignancies.

To directly evaluate the immunogenicity of CMC microneedle arrayincorporated antigens, the well characterized model antigen ovalbuminwas used. Pyramidal arrays were fabricated incorporating either solubleovalbumin (sOVA), particulate ovalbumin (pOVA), or arrays containingboth pOVA along with CpGs. The adjuvant effects of CpGs are wellcharacterized in animal models, and their adjuvanticity in humans iscurrently being evaluated in clinical trials.

Immunization was achieved by applying antigen containing CMC-microneedlearrays to the ears of anesthetized mice using a spring-loaded applicatoras described above, followed by removal of the arrays 5 minutes afterapplication. These pyramidal microneedle arrays contained about 5 wt %OVA in CMC and about 0.075 wt % (20 μM) CpG. As a positive control, genegun based genetic immunization strategy using plasmid DNA encoding OVAwas used. Gene gun immunization is among the most potent andreproducible methods for the induction of CTL mediated immune responsesin murine models, suggesting its use as a “gold standard” for comparisonin these assays.

Mice were immunized, boosted one week later, and then assayed forOVA-specific CTL activity in vivo. Notably, immunization with arrayscontaining small quantities of OVA and CpG induced high levels of CTLactivity, similar to those observed by gene gun immunization (FIG. 27).Significant OVA-specific CTL activity was elicited even in the absenceof adjuvant, both with particulate and soluble array delivered OVAantigen. It is well established that similar responses requiresubstantially higher doses of antigen when delivered by traditionalneedle injection.

To evaluate the stability of fabricated arrays, batches of arrays werefabricated, stored, and then used over an extended period of time. Asshown in FIG. 28, no significant deterioration of immunogenicity wasobserved over storage periods spanning up to 80 days (longest time pointevaluated). Thus, the CMC microneedle arrays and this deliverytechnology can enable effective cutaneous delivery of antigen andadjuvants to elicit antigen specific immunity.

To evaluate the delivery of a biologically active small molecule,pyramidal CMC-microneedle arrays were fabricated with the low molecularweight chemotherapeutic agent Cytoxan® (cyclophosphamide), or withFluoresBrite green fluorescent particles as a control. Cytoxan® wasintegrated at a concentration of 5 mg/g of CMC, enabling delivery ofapproximately about 140 μg per array. This is a therapeutically relevantconcentration based on the area of skin targeted, yet well below levelsassociated with systemic toxicities. Living human skin organ cultureswere used to assess the cytotoxicty of Cytoxan®. Cytoxan® was deliveredby application of arrays to skin explants as we previously described.Arrays and residual material were removed 5 minutes after application,and after 72 hours of exposure, culture living skin explants werecryo-sectioned and fixed. Apoptosis was evaluated using greenfluorescent TUNEL assay (In Situ Cell Death Detection Kit, TMR Green,Roche, cat#:11-684-795-910). Fluorescent microscopic image analysis ofthe human skin sections revealed extensive apoptosis of epidermal cellsin Cytoxan® treated skin as shown in FIG. 29A. As shown in FIG. 29B, novisible apoptosis was observed in fluorescent particle treated skinthough these particles were evident, validating that the observed areawas accurately targeted by the microneedle array.

Direct Fabricated Microneedle Arrays

The micromilling of mastermolds described above allows the production ofmicroneedle arrays with a variety of geometries. In another embodiment,systems and methods are provided for fabricating a microneedle array bydirectly micromilling various materials, such as dried CMC sheets. Thesame general tooling that was described above with respect to themicromilling of mastermolds can be used to directly micromillingmicroneedle arrays.

Direct micromilling of microneedle arrays eliminates the need formolding steps and enables a simplified, scalable, and preciselyreproducible production strategy that will be compatible with largescale clinical use. Moreover, direct fabrication of the microneedlearrays through micromilling enables greater control of microneedlegeometries. For example, micromilling permits the inclusion ofmicroneedle retaining features such as undercuts and/or bevels, whichcannot be achieved using molding processes.

The reproducibility of direct milling of microneedle arrays isparticular beneficial. That is, in direct micromilling all of themicroneedles are identical as a result of the milling fabricationprocess. In molding operations, it is not uncommon for some needles tobe missing or broken from a given patch as a result of the process ofphysically separating them from the molds. For use in certain medicalapplications, the reproducibility of the amount of bioactive componentsin the array is very important to provide an appropriate level of“quality control” over the process, since irregularities in the needlesfrom patch to patch would likely result in variability in the dose ofdrug/vaccine delivered. Of course, reproducibility will also be animportant benefit to any application that requires FDA approval.Spincast/molded patches would require special processes to assureacceptable uniformity for consistent drug delivery. This quality controlwould also be likely to result in a certain percentage of the patches“failing” this release test, introducing waste into the productionprocess. Direct micromilling eliminates or at least significantlyreduces these potential problems.

Molding processes also have inherent limitations because of the need tobe able to fill a well or concavity and remove the cured molded partfrom that well or concavity. That is because of mold geometries,undercuts must generally be avoided when molding parts or the part willnot be removable from the mold. That is, a geometrical limitation of amolded part, such as a molded microneedle array, is that any featurelocated closer to the apex must be narrower than any feature locatedtoward the base.

Accordingly, in view of these limitations, FIG. 20 illustrates schematicrepresentation of microneedle shapes and structures that are generallysuitable for fabrication by molding. That is, the shapes and structuresshown in FIG. 20 do not contain any undercuts that would prevent thepart (i.e., the microneedles) from being removed from a production mold.In contrast, FIG. 30 illustrates a beveled, undercut microneedle shapethat cannot be molded in the manners described herein.

This geometry can only be created through direct fabrication using theproposed micromilling technology. The negative (bevel) angle facilitatesbetter retention of the microneedles in the tissue. In addition, becausethe microneedle of FIG. 30 has a wider intermediate portion (with alarger cross-sectional dimension) above a lower portion (with a smallercross-sectional dimension), a greater amount of the bioactive materialcan be delivered by configuring the microneedle to hold or store thebioactive material in the wider section, which is configured to beretained within the skin. Thus, the larger cross-sectional dimension ofthe intermediate portion can “carry” the bulk of the bioactivecomponent. Since the lower portion tapers to a narrower cross-sectionaldimension, the wider intermediate portion will obtain good penetrationfor delivery of the bioactive component into the skin layer. A portionabove the intermediate portion desirably narrows to a point tofacilitate entry of the microneedles into the skin layers.

Another limitation of molded parts is that it can be difficult toprecisely fill a very small section of a mold. Since production moldsfor microneedle arrays comprise numerous very small sections, it can bedifficult to accurately fill each well. This can be particularlyproblematic when the mold must be filled with different materials, suchas a material that contains a bioactive component and a material thatdoes not contain a bioactive component. Thus, if the production mold isto be filled with layers, it can be difficult to accurately fill thetiny wells that are associated with each microneedle. Suchreproducibility is particularly important, since the microneedles areintended to deliver one or more bioactive components. Thus, even slightvariations in the amounts of bioactive component used to fill productionmolds can be very undesirable.

Also, by using a lamination structure to form a sheet or block that canbe micromilled, various active components can be integrated into asingle microneedle by vertical layering. For example, in an exemplaryembodiment, CMC-hydrogel and CMC-sOVA-hydrogel (80% CMC/20 wt % OVA)were layered into the form of a sheet or block. This composite sheet canbe micro-machined using the direct micromilling techniques describedherein.

FIG. 31 is a stereo-microscopic image analysis of an entire microneedlearray. The microneedle comprises a 10×10 array of microneedles. FIG. 32is an enlarged segment of the microneedle array of FIG. 31. The layeringof two components is shown in FIG. 32, which illustrates darker areas ofthe microneedles at tip portions and lighter areas of the microneedlesat base portions. The darker layer at the tip represents the layercomprising a bioactive component, in this case soluble ovalbumincontained in a CMC layer.

Although the formation of a layer containing active material (e.g.,antigen) and the subsequent micromilling of the layer (and any otheradjacent layers) may require the use of relatively large amounts of theactive material, the material can be removed (e.g., in the form ofchips), recovered, and recycled. Direct machining technology is notrestricted by the geometrical constraints arising from themolding/de-molding approach, and thus, is capable of creating moreinnovative needle designs (e.g., FIG. 30), which can significantlyimprove the retained needle-volume and needle retention time in theskin.

The production of sheets or blocks by forming a plurality of layers canprovide a solid material that can be micro-machined and which cancomprise one or more layers with a bioactive component. For example, adissoluble solid carboxymethylcellulose polymer based block or sheetwith well-defined and controlled dimensions can be fabricated by alamination process. The resulting sheet or block can be fullymachinable, similar to the machining of plastic or metal sheets orblocks. As described herein, the fabrication process can be suitable forthe incorporation of bioactive components into the matrix withoutsignificantly reducing their activity levels.

As described below, a fabricated sheet of material (such as a CMC basedmaterial) can be directly micro-machined/micromilled) to produce one ormore microneedle arrays suitable for delivering active ingredientsthrough the skin. This dissoluble biocompatible CMC block-material canbe used for the delivery of soluble or insoluble and particulate agentsin a time release manner for body surface application.

The biocompatible material can be suitable for implants in deeper softor hard tissue when dissolution of the scaffolding material is requiredand useful.

The following method can be used to prepare a carboxymethylcellulose(CMC) polymer low viscosity hydrogel to 12.5% concentration. The 12.5%carboxymethylcellulose (CMC) low viscosity hydrogel can be prepared inwater or other biocompatible buffer, such as (but not limited to) PBS orHBS. During the preparation of the polymer solution, soluble agents(such as nucleic acid, peptides, proteins, lipids or other organic andinorganic biologically active components) and particulates can be added(e.g. ovalbumin, a soluble agent). Ferrous particulates carrying activeingredients at 20 w/w % of CMC can be used.

The preparation of 1000 g sterile 12.5% CMC hydrogel with no activecomponent can be achieved as follows:

1) Measure 125 g CMC, add 875 g water or other water based solvent.

2) Stir to homogeneity in overhead mixer.

3) Autoclave homogenate to sterility at 121 degrees Celsius for 1 hour(the autoclaving step can reduce viscosity for improved layering)

4) Cool to 22 degrees Celsius.

5) Vacuum treat the resulting material at 10 torr and 22 degrees Celsiusfor 1 hour to remove trapped micro-bubbles.

6) Centrifuge product at 25,000 g for 1 hour in vacuum chamberedcentrifuge (for floating and further removing residual micro bubbles).

7) Store the CMC-hydrogel product at 4 degrees Celsius.

The preparation of 1000 g sterile 12.5 w/w % dry content 20/80%ovalbumin/CMC hydrogel can be achieved as follows:

1) Measure 100 g CMC add 650 g water or other water based solvent.

2) Stir to homogeneity in overhead mixer.

3) Autoclave homogenate to sterility at 121 degrees Celsius for 1 hour(this autoclaving step can reduce viscosity for improved layering).

4) Cool to 22 degrees Celsius.

5a) Dissolve 25 g ovalbumin in 225 g water.

5b) Sterile filter ovalbumin solution on 0.22 μm pore sized filter.

6) Mix to homogeneity, under sterile conditions the 750 g CMC hydrogelwith 250 g sterile ovalbumin solution.

7) Vacuum treat the resulting material at 10 torr and 22 degrees Celsiusfor 1 hour to remove trapped micro-bubbles.

8) Centrifuge product at 25,000 g for 1 hour in vacuum chamberedcentrifuge (for floating and further removing residual micro bubbles).

9) Store the CMC-hydrogel product at 4 degrees Celsius.

The preparation of 100 g sterile 12.5 w/w % dry content 20/80%particulate-ovalbumin/CMC hydrogel can be achieved as follows:

1) Measure 10 g CMC add 87.5 g water or other water based solvent.

2) Stir to homogeneity in overhead mixer.

3) Autoclave homogenate to sterility at 121 degrees Celsius for 1 hour(this autoclaving step can reduce viscosity for improved layering).

4) Cool to 22 degrees Celsius.

5) Disperse 2.5 g particulate-ovalbumin in the 97.5 g, 22 degreesCelsius CMC-hydrogel and mix to homogeneity, under sterile conditions.

6) Vacuum treat the resulting material at 10 torr and 22 degrees Celsiusfor 2 hour to remove trapped micro-bubbles.

7) Centrifuge product at 3,000 g for 1 hour in vacuum chamberedcentrifuge (for floating and further removing residual micro bubbles).

8) Store the CMC-hydrogel product at 4 degrees Celsius.

Note in this example, particulate-ovalbumin is prepared from activatediron beads reaction to ovalbumin. However, it should be noted that theabove descriptions are only exemplary embodiments and other compoundsand active ingredients can be used.

A solid block/sheet carboxymethylcellulose (CMC) can be fabricated inthe following manner using the low viscosity CMC-hydrogels describedabove.

The fabrication process can comprise a laminar spreading of the polymerat a defined thickness and a drying of the layered polymer to less thanabout 5% water content using sterile dried air flow over the surface ofthe polymer layer. The above two acts can repeated until the desiredblock thickness is achieved.

A method of performing a laminar CMC-hydrogel layering of a definedthickness over the casting mold assembly is described with reference toFIG. 33. FIG. 33 illustrates a cross-sectional view of the casting-moldassembly which includes: (a) casting bed; (b) adjustable casting bedwall; (c) casting-bed depth adjustment assembly; and (d) an acrylicspreader. It should be noted that FIG. 33 is not drawn to scale orotherwise shown with elements in their proper proportions.

The casting mold assembly can be constructed from acrylic (Plexiglas)and can comprise a casting bed base unit, a vertically adjustablehydrophobic casting-bed wall, and a casting-bed adjustment mechanism.The casting bed base unit (a1) can include a removable/replaceablecasting bed top plate (a2) with an attached cellulose layer (a3). Thecellulose layer can be about 0.5 mm in thickness. The verticallyadjustable hydrophobic casting-bed wall (b) can be adjusted using thecasting-bed depth adjustment mechanism, which can be comprised oflead-screw (c1) and level adjustment knob (c2). In the illustratedembodiment, a quarter turn of this knob can result in a 0.5 mm lift ofthe bed wall.

Initially, the adjustable casting bed wall can be set to height wherethe distance between the acrylic spreader and the cellulose layer of thebed is about 1 mm when the spreader is in position. A predefined volume(e.g., about 0.1 ml/cm2) of the 12.5% CMC-hydrogel can be added andlayered. The layer can be evened or leveled by sliding the acrylicspreader (d) on the top surface of the adjustable casting wall to yieldan even layer of about 1 mm of CMC-hydrogel. The layered CMC-hydrogelcan be dried to a solid phase in the drying apparatus shown in FIG. 34and described in more detail below.

The layering and drying steps can be repeated until the desired layeredstructure (sheet) is achieved. The casting bed wall can be raised by anappropriate amount during the addition of each layer. For example, afteradding each layer, the bed wall can be raised or lifted by about 0.5 mmThus, the above-described cycle can deposit about 0.5 mm solid CMClayer. The process (e.g., the layering of material, the raising of bedwall, etc.) can be repeated until the desired block thickness achieved.

The layered CMC-hydrogel polymer can be dried in various manners. Forexample, FIG. 34 illustrates a drying apparatus that can be used to drythe various deposited layers of the sheet material. It should be notedthat FIG. 34 is not drawn to scale or otherwise shown with elements intheir proper proportions. A fan can provide continuous gas flow (e.g.,air or other inert gas, such as nitrogen) over the CMC-hydrogel layeredin the casting mold assembly. The gas flow will result in a gentledehydration of the CMC-hydrogel layer. The drying speed can be adjustedto prevent or reduce gas enclosures (e.g., air bubbles) in the solid CMCproduct. The humid air over the layer can be dried over desiccant (e.g.,an air dryer or dehumidifier), temperature adjusted, and returned overthe hydrogel again by the speed-controlled fan. A hygrometer can bepositioned on the humid side of the chamber to provide an indication ofthe status of the drying process. After a predetermined dryness has beenachieved, as indicated by the hygrometer, the drying process can beended.

Airflow can be adjusted to affect the drying speed. In the exemplaryembodiment, the airflow is controlled to be between about 0.1-2.0 m/sec;the temperature is between ambient and about 50 degrees Celsius. Usingthese configurations, the drying time of a single layer CMC-hydrogel canbe about 0.5-4 hours depend on the airflow and the set temperature.

The pure CMC based product can be transparent, light off white, or ambercolored. Its specific gravity can be about 1.55-1.58 g/ml. The productis desirably free of micro-bubbles and otherwise suitable forfabricating micron scale objects. The physical characterization of thefinal block/sheet product (hardness, tensile strength, etc.) can vary,but should generally be able to resist physical stresses associated withmicromilling.

As described above, the microneedle arrays disclosed herein are capableof providing reliable and accurate delivery methods for variousbioactive components. The structural, manufacturing, and distributionadvantages characteristic of the above-described microneedle arrays canbe particularly applicable for use in delivering vaccines. Advantages ofthese microneedle arrays include (1) safety, obviating the use ofneedles or living vectors for vaccine delivery, (2) economy, due toinexpensive production, product stability, and ease of distribution, and3) diversity, via a delivery platform compatible with diverse antigenand adjuvant formulations.

Moreover, cutaneous immunization by microneedle array has importantadvantages in immunogenicity. The skin is rich in readily accessibledendritic cells (DCs), and has long been regarded as a highlyimmunogenic target for vaccine delivery. These dendritic cellpopulations constitute the most powerful antigen presenting cells (APCs)identified thus far. For example, genetic immunization of skin resultsin transfection and activation of dendritic cells in murine and humanskin, and these transfected dendritic cells synthesize transgenicantigens, migrate to skin draining lymph nodes, and efficiently presentthem through the MHC class I restricted pathway to stimulate CD8+T-cells. The immune responses induced by skin derived DCs are remarkablypotent and long-lasting compared to those induced by other immunizationapproaches. Recent clinical studies demonstrate that even conventionalvaccines are significantly more potent when delivered intradermally,rather than by standard intramuscular needle injection. Thus,microneedle arrays can efficiently and simultaneously deliver bothantigens and adjuvants, enabling both the targeting of DCs and adjuvantengineering of the immune response using the same delivery platform.

Cancer Therapy Applications

Bioactive components used with the microneedle arrays described hereincan include one or more chemotherapeutic agents. Effective and specificdelivery of chemotherapeutic agents to tumors, including skin tumors isa major goal of modern tumor therapy. However, systemic delivery ofchemotherapeutic agents is limited by multiple well-establishedtoxicities. In the case of cutaneous tumors, including skin derivedtumors (such as basal cell, squamous cell, Merkel cell, and melanomas)and tumors metastatic to skin (such as breast cancer, melanoma), topicaldelivery can be effective. Current methods of topical delivery generallyrequire the application of creams or repeated local injections. Theeffectiveness of these approaches is currently limited by limitedpenetration of active agents into the skin, non-specificity, andunwanted side effects.

The microneedle arrays of the present disclosure can be used as analternative to or in addition to traditional topical chemotherapyapproaches. The microneedle arrays of the present disclosure canpenetrate the outer layers of the skin and effectively deliver theactive biologic to living cells in the dermis and epidermis. Delivery ofa chemotherapeutic agents results in the apoptosis and death of skincells.

Further, multiple bioactive agents can be delivered in a singlemicroneedle array (patch). This enables an immunochemotherapeuticapproach based on the co-delivery of a cytotoxic agent with and immunestimulant (adjuvants). In an immunogenic environment created by theadjuvant, tumor antigens releases from dying tumor cells will bepresented to the immune system, inducing a local and systemic anti-tumorimmune response capable of rejecting tumor cells at the site of thetreatment and throughout the body.

Example 6

In an exemplary embodiment, the delivery of a biologically active smallmolecule was studied. In particular, the activity of thechemotherapeutic agent Cytoxan® delivered to the skin with CMCmicroneedle arrays was studied. The use of Cytoxan® enables directmeasurement of biologic activity (Cytoxan® induced apoptosis in theskin) with a representative of a class of agents with potential clinicalutility for the localized treatment of a range of cutaneousmalignancies.

To directly evaluate the immunogenicity of CMC microneedle arrayincorporated antigens, the well characterized model antigen ovalbuminwas used. Pyramidal arrays were fabricated incorporating either solubleovalbumin (sOVA), particulate ovalbumin (pOVA), or arrays containingboth pOVA along with CpGs. The adjuvant effects of CpGs are wellcharacterized in animal models, and their adjuvanticity in humans iscurrently being evaluated in clinical trials.

Immunization was achieved by applying antigen containing CMC-microneedlearrays to the ears of anesthetized mice using a spring-loaded applicatoras described above, followed by removal of the arrays 5 minutes afterapplication. These pyramidal microneedle arrays contained about 5 wt %OVA in CMC and about 0.075 wt % (20 μM) CpG. As a positive control, genegun based genetic immunization strategy using plasmid DNA encoding OVAwas used. Gene gun immunization is among the most potent andreproducible methods for the induction of CTL mediated immune responsesin murine models, suggesting its use as a “gold standard” for comparisonin these assays.

Mice were immunized, boosted one week later, and then assayed forOVA-specific CTL activity in vivo. Notably, immunization with arrayscontaining small quantities of OVA and CpG induced high levels of CTLactivity, similar to those observed by gene gun immunization.Significant OVA-specific CTL activity was elicited even in the absenceof adjuvant, both with particulate and soluble array delivered OVAantigen. It is well established that similar responses requiresubstantially higher doses of antigen when delivered by traditionalneedle injection.

To evaluate the stability of fabricated arrays, batches of arrays werefabricated, stored, and then used over an extended period of time. Nosignificant deterioration of immunogenicity was observed over storageperiods spanning up to 80 days (longest time point evaluated). Thus, theCMC microneedle arrays and this delivery technology can enable effectivecutaneous delivery of antigen and adjuvants to elicit antigen specificimmunity.

To evaluate the delivery of a biologically active small molecule,pyramidal CMC-microneedle arrays were fabricated with the low molecularweight chemotherapeutic agent Cytoxan® (cyclophosphamide), or withFluoresBrite green fluorescent particles as a control. Cytoxan® wasintegrated at a concentration of 5 mg/g of CMC, enabling delivery ofapproximately about 140 μg per array. This is a therapeutically relevantconcentration based on the area of skin targeted, yet well below levelsassociated with systemic toxicities. Living human skin organ cultureswere used to assess the cytotoxicty of Cytoxan®. Cytoxan® was deliveredby application of arrays to skin explants as we previously described.Arrays and residual material were removed 5 minutes after application,and after 72 hours of exposure, culture living skin explants werecryo-sectioned and fixed. Apoptosis was evaluated using greenfluorescent TUNEL assay (In Situ Cell Death Detection Kit, TMR Green,Roche, cat#:11-684-795-910). Fluorescent microscopic image analysis ofthe human skin sections revealed extensive apoptosis of epidermal cellsin Cytoxan® treated skin. No visible apoptosis was observed influorescent particle treated skin though these particles were evident,validating that the observed area was accurately targeted by themicroneedle array.

Example 7

In another embodiment, topical treatment of established tumors withdoxorubicin and/or Poly(I:C) integrated into MNAs established tumorregression and durable immunity that can protect from subsequent lethalsystemic tumor challenges.

Novel therapeutic approaches for treating established skin tumors wereprovided based on the combined effect of MNA delivered chemotherapy, MNAdelivered immunostimulant therapy, and/or MNAs delivering combinationchemo-immunotherapy. The B16 melanoma model was used as a model tumor totest these novel approaches. The B16 melanoma model is very wellstudied, and is one of the most aggressive murine skin cancers. Of allskin tumor models available, an established B16 tumor is among the mostdifficult to treat. Further, B16 has a very high metastatic potential,enabling a clinically relevant assessment of systemic tumor immunity.

B16 skin tumors were established in normal mice by injection. Visibleestablished cutaneous tumors were treated once weekly for three weekswith MNAs containing either doxorubicin alone, Poly(I:C) alone, ordoxorubicin and Poly(I:C) incorporated into the same MNA. Thedoxorubicin dose chosen corresponds to an MNA dose that inducesapoptosis in human skin without causing necrosis. Tumor growth andsurvival were measured regularly for the duration of the study.Treatment with MNAs containing doxorubicin alone slowed tumor growth,and improved survival (30%) compared to that observed in untreated tumorbearing animals that had a 100% mortality rate. Further, treatment withMNAs containing Poly(I:C) alone slowed tumor growth, and improvedsurvival (50%) compared to that observed in untreated tumor bearinganimals that had a 100% mortality rate. Remarkably, treatment withcontaining both doxorubicin+Poly(I:C) substantially slowed tumor growthin all animals, and eradicated tumors completely in 8 out of 10 mice.This was reflected in 80% long term survival extending through day 70.

Surviving animals were evaluated to determine whether they developedlong-term immunity against these same tumors. Specifically, systemicimmunity was evaluated in these animals, including the durability of theimmune response and the capacity of surviving animals to survive IVchallenge. In particular, sixty days after the initial MNA treatment,mice were treated with a lethal dose of B16. Fourteen days later, micewere sacrificed and lung metastases were quantified microscopically.Treated mice demonstrated dramatically reduced numbers of lung lesionscompared to naïve controls. Taken together, these results demonstratethe capacity of MNAs to deliver chemotherapeutic agents, immunestimulants, and combinations of these agents to both induce regressionof established skin tumors, and to simultaneously induce durablesystemic tumor specific immune responses capable of protecting thesubject from subsequent tumors.

In another embodiment, Poly-ICLC can be substituted for Poly(I:C), andMNAs can be formed, for example, with Poly-ICLC in combination with atleast one other chemotherapeutic agent (e.g., doxorubicin).

As discussed above, the one or more chemotherapeutic agents can includeone or more immunostimulants agents (specific and non-specific) known bythose skilled in the art to stimulate the immune system to reject anddestroy tumors, such as Poly(I:C) and Poly-ICLC. These immunostimulantscan be integrated into the MNAs along with other chemotherapeuticagents, such as cytotoxic agents like doxorubicin Immunostimulants thatcan be used in the manner described herein include adjuvants, toll-likereceptors (TLRs), ribonucleotides and deoxyribonucleotides, doublestranded RNAs (dsRNA), and derivatives of Poly(I:C).

Compositions Comprising Bioactive Components and Methods of Forming theSame

As discussed in detail above, dissolvable microneedle arrays can be usedfor transdermal delivery of drugs and biologics to human skin. Suchmicroneedle arrays can include one or more bioactive components,including drugs, adjuvants, antigens, and chemotherapeutic agents suchas Doxorubicin.

In some embodiments, one or more bioactive molecules can be linked tocarboxymethylcellulose or similar biocompatible components. Themethodology for chemically combining these agents can include methodsthat create a linkage designed to release one or more active componentsin target microenvironments by utilizing unique features of themicroenvironment. This can include, for example, the acidic environmentof a cellular compartment or vesicle, or the reducing environment of atumor. In another embodiment of this invention this can include thecombined delivery of carboxymethylcellulose conjugate and an agentfacilitating cleavage of the conjugate that releases an activecomponent. Delivery of the release facilitating agent can besimultaneous or sequential with delivery of the conjugate.

Advantages of providing cleavable bioactive components include thecapability to deliver bioactive components in a protected fashion,limiting drug release to the target site thereby enhancing effectivedelivery concentrations while minimizing systemic or non-specificexposures. Further, in the event that the bioactive component is atargeting entity, drug release can be targeted to specific cell types orcells with certain metabolic features. A further advantage is thepotential for protracted or sustained release delivery.

Carboxymethylcellulose or similar biocompatible components can beselected to enable fabrication into dissolvable microneedle arrays suchas the arrays and methods of fabrication described herein.Alternatively, these conjugates can be delivered into the body by othermeans such as needle injection or ingestion.

As described herein, molecules of bioactive components, such aspharmaceutically active compounds, can be chemically conjugated tocarboxymethylcellulose. In some embodiments, this is achieved using acleavable bond capable of releasing the active chemical moiety incertain biologically natural or engineered environments. This technologycan be useful for controlled and targeted drug delivery. Further, due tostructural features of CMC, CMC-drug conjugates can be delivered bytraditional methods including needle injection, and by novel deliverystrategies by physically hardening the conjugate into solid structuresthat can be implanted, or that can serve as a combination drug/deliverydevice in the same entity. Examples of the latter would include CMC-drugconjugates fabricated into dissolvable microneedle arrays.

The example presented below includes a chemotherapeutic agent,Doxorubicin, which can be chemically linked to carboxymethylcellulosethrough a cleavable disulfide bond. However, a cleavable disulfide bondis just one chemical linkage strategy that can be used to link moleculesof a bioactive component to a substrate, such as a CMC substrate. Forexample, in addition to disulfide bonds, other chemical linkagestrategies that can be used, so long as they are cleavable in theintended environment, include crosslinking and chemical modificationusing primary amines (—NH₂), carboxyls (—COOH), and carbonyls (—CHO).

As discussed below, this approach can be chemically compatible with abroad range of other bioactive components. Further, other known chemicallinkage strategies could be utilized to conjugate a broad range ofchemicals/drugs to CMC, including small molecule drugs, peptide andprotein drugs. These drugs can be linked to a CMC substrate singly or incombinations, and in the presence or absence of one or more targetingmolecules.

Example 6

In this example, Doxorubicin is chemically linked tocarboxymethylcellulose (CMC) through a cleavable disulfide bond. Thesynthesis strategy employed creates a sulfhydryl-bridged doxorubicin-CMCcomplex that is cleavable (i.e., able to release the drug) in areductive environment such as cytosol and other cell-compartments, theextracellular space of the tumor microenvironment, or reducingenvironments created by cellular stress (redox). Further, the release ofdoxorubicin could also be triggered by targeted introduction of reducingagent such as dithiothreitol (DTT), beta-mercaptoethanol (MEA),Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or others, togetherwith or subsequent to drug delivery. FIG. 38 shows a chemical schematicof the Doxorubicin-S—S-CMC construct.

In this example the synthesis process is composed of 3 major steps:

1) Highly purified Doxorubicin-SH preparation by 3′ amin-conversion tosulfhydryl-group.

2) Amination of free HO-groups on dextrose units of CMC.

3) Crosslinking of Doxorubicin-SH to NH₂-CMC

Detailed approaches for achieving the three above steps are providedbelow.

(1) Highly Purified Doxorubicin-SH Preparation by 3′ Amin-Conversion toSulfhydryl-Group.

The process relies on linking doxorubicin to a solid support throughsulfhydryl-bridge formation. After complete removal of the residualreactants the doxorubicin-SH is cleaved off of the support and releasedusing a reductive agent (e.g., MEA). The eluted doxorubicin-SH is vacuumdried to remove the reducing agent and stored at −20° C. orreconstructed for further use. The described process ensures that onlypure modified sulfhydryl-doxorubicin is recovered as final product.

Methods:

a. Preparation of NH₂-Cellulose for Solid Support Using Epichlorohdrinand Ammonium Hydroxide

FIG. 39 shows a two-step ammination of cellulose in alkaline environmentusing epichlorohydrinand ammonium hydroxide.

-   -   Rehydrate 25 g cellulose particles in 200 ml 2n NaOH.    -   With continuous stirring bring it to 60° C.    -   When the cellulose suspension reached 60° C. 1.5 g of        epichlorohdrin per g cellulose is added.    -   Vigorously stir at 60° C. for 2 hours.    -   Wash the epoxide-cellulose 4× with 500 ml distilled water to        obtain pH 7-8.    -   Resuspend epoxide-cellulose particles in 200 ml 0.1n NaOH.    -   With continuous stirring bring it to 60° C.    -   When the epoxide-cellulose suspension reached 60° C., 150 ml        ccNH₄—OH is added.    -   Vigorously stir at 60° C. for 2 hours.    -   Wash the aminated-cellulose 4× with 500 ml distilled water to        obtain pH ˜7.    -   Store at 4° C. until used in (b) doxorubicin/NH₂-cellulose        crosslinking reaction.

b. Crosslinking Doxorubicin to Aminated-Cellulose Using InternallyCleavable Dithiobis[Succinimidyl Propionate] (DSP) Adapter

FIG. 40 shows Doxorubicin S—S cross-linked to cellulose support, A:Doxorubicin-S—S-cellulose after 4 cycles of washing, B:Aminated-cellulose with no doxorubicin binding from the sham reactionafter 4 washing cycles.

-   -   Prepare 1 g NH₂-cellulose in 10 ml PBS    -   Prepare 8 ml doxorubicin solution in water at 1 mg/ml    -   Prepare 80 mg DSP in 2 ml dry DMSO    -   Mix all 3 reagents and incubate at RT for 30 min    -   Prepare sham reaction with DMSO only, no DSP.    -   Wash doxorubicin-S—S-cellulose conjugate 4× with 50 ml water pH        adjusted to 5 to remove unbound doxorubicin and other residual        reactants.    -   After final wash resuspend cleaned doxorubicin-cellulose in 10        ml PBS, store at 4° C. until desired cleavage of the S—S bonds        and release of the doxorubicin-SH.

c. Elution and Purification of Clean Doxorubicin-SH from CelluloseSupport

FIG. 41 shows Doxorubicin-S—S-cellulose (#4) and cellulose sample fromsham reaction (#5) prepared for cleavage and elution in a column (A).The doxorubicin-SH (B) after elution was further purified byvacuum-drying and reconstructed in water.

-   -   One ml samples of doxorubicin-S—S-cellulose slurry (FIG. 40A)        and the sham control (FIG. 40B) were packed in        chromatography-columns (FIG. 41A).    -   Columns were washed with 1 ml distilled water.    -   To cleave and elute the doxorubicin-SH 0.5 ml of 0.1 M        2-mercaptoethanol was added.    -   Columns were incubated at 37° C. for 30 min and then eluted.    -   The elution was repeated with an additional 0.5 ml 0.1 M        2-mercaptoethanol.    -   The collected doxorubicin-SH was vacuum dried.    -   Dried doxorubicin-SH was stored at −20° C. desiccated or        reconstructed in H₂O for further use (FIG. 41B).        (2) Amination of Free HO-Groups on Dextrose Units of CMC.

The basic reactions of the amination of CMC are performed as describedabove but in solution. Therefore the residual reactants are removed byrepeated precipitation with ethanol since CMC is generally insoluble inorganic solvents.

FIG. 42 shows a two-step ammination of CMC in alkaline environment usingepichlorohydrinand ammonium hydroxide.

Methods:

a. Prepare 5% CMC in H₂O

-   -   dissolve 20 g in 400 ml    -   On a heating/stirring plate bring it to 60° C.    -   Move it to an oven equipped with a stirring plate and set to 60°        C.    -   Stir 0/N, completely dissolve CMC.

b. NH₂-CMC Preparation Using Epichlorohdrin and Ammonium Hydroxide.

-   -   To 200 ml 5% CMC solution add NaOH to bring it to final        concentration of 2.5 n.    -   With continuous stirring bring it to 60° C.    -   When the CMC solution reaches 60° C. 1.5 g, epichlorohdrin per g        CMC is added    -   Vigorously stir at 60° C. for 2 hours.    -   Add epichlorohydrin (18 mmol/g CMC that is 1.66 g/g CMC or 1.4        ml/g CMC).        Temperature will rise to 65−70° C., let it cool down to 60° C.    -   Reaction time is 2 h from the addition of epichlorohydrin.    -   Precipitate Epoxide-CMC w/EtOH by adding 4 vol. (80% final        conc.) 0/N at 4° C.    -   Centrifuge at 2K rpm for 20 min, decant, air-dry briefly.    -   Resolve Epoxide-CMC in 200 ml 0.1n NaOH    -   On a heating/stirring plate bring it to 60° C.    -   Add 150 ml ccNH₄—OH (29% w/v)    -   React for 2 h at 60° C.    -   Precipitate as in steps above.    -   Wash pelleted NH₂-CMC w/90% EtOH twice.    -   Resolubilize in 100 ml H₂O (get it in solution completely).    -   Repeat precipitation step as before.    -   Reconstruct in 100 ml H₂O.    -   Neutralize the residual NaOH and NH₄—OH with 4 n HCl, get ˜pH        6.5-8.5 range.    -   Test for recovery efficiency of NH₂-CMC. (Expected is 60-80%)

3) Cross-Linking of Doxorubicin-SH to NH₂-CMC.

The cross-linking of Doxorubicin-SH to NH₂-CMC utilizes ahetero-bi-functional adapter (N-Succinimidyl3-(2-pyridyldithio)-propionate (SPDP)) to achieve a short extension atthe 3′-NH₂ of the doxorubicin upon release from the doxorubicin-S—S-CMCconjugate preserving the functionality of doxorubicin.

Methods:

-   -   Prepare 1 g NH₂-CMC in 10 ml PBS    -   Prepare 5 ml doxorubicin-SH solution in water at 0.5 mg/ml    -   Prepare 40 mg SPDP in 1 ml dry DMSO    -   Mix all 3 reagents and incubate at RT for 30 min    -   Prepare sham reaction with DMSO only, no SPDS.    -   Precipitate doxorubicin-S—S-CMC conjugate 4× volume of ethanol        pH adjusted to 5 with 1 n HCl to remove unbound doxorubicin and        other residual reactants.    -   Resolve pelleted doxorubicin-S—S-CMC in 10 ml water.    -   Dialyze doxorubicin-S—S-CMC solution using spectrapore dialysis        tubing (MW cutoff 3,500 dalton) against 21 distilled water        changing the distilled water 2× in every 12 hours at 4° C.    -   Precipitate doxorubicin-S—S-CMC as above with ethanol    -   Vacuum dry pelleted doxorubicin-S—S-CMC.    -   Store at −20° C. until use or reconstruct at the required        concentration with water.

Conjugation and release were validated by quantification of the activeepoxide group using titration according to equation:

Other Linkable Bioactive Components

Although Doxorubicin is the bioactive component in the above-disclosedembodiment, other bioactive components can be used and be linked to aCMC or other biocompatible structural substrate. Bioactive componentssuitable for use include other cytotoxic agents traditionally used totreat cancer. Such agents may include, but are not limited to,alkylating agents such as busulfan, hexamethylmelamine, thiotepa,cyclophosphamide (Cytotaxan), mechlorethamine, uramustine, melphalan,chlorambucil, carmustine, streptozocin, dacarbazine, temozolomide,ifosfamide, and the like; anti-metabolites such as methotrexate,azathioprine, mercaptopurine, fludarabine, 5-fluorouracial, and thelike; anthracyclines such as daunorubicin, epirubicin, idarubicin,mitoxantrone, and the like; plant alkaloids and terpenoids such asvincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, doclitaxel, and the like; topoisomerase inhibitors such asirinotecan, amsacrine, topotecan, etoposide, teniposide, and the like;antibody agents, such as rituximab, trastuzumab, bevacizumab, erlotinib,dactinomycin; finasteride; aromatase inhibitors; tamoxifen; goserelin;imatinib mesylate.

Other suitable compounds that can be the bioactive components usedherein include, for instance, proteinaceous compounds, such as insulin,peptide antimicrobials (e.g., naturally occurring defensins,cathelicidins and other proteins with anti-bacterial and/or antiviralactivity and synthetic derivatives of naturally occurring peptideantimicrobials including truncated or structurally modified variants),immunoglobulins (e.g., IgG, IgM, IgA, IgE), TNF-α, antiviralmedications, etc.; polynucleotide agents, such as plasmids, siRNA, RNAi,nucleoside anticancer drugs, vaccines, etc.; small molecule agents, suchas alkaloids, glycosides, phenols, etc.; anti-infection agents,hormones, drugs regulating cardiac action or blood flow, pain control;and so forth. Suitable compounds also include electrophilic nitro-fattyacids (FA-NO₂) such as nitro-oleic acid (OA-NO₂) and nitro-linoleic acid(LN—NO₂) and their derivatives. Suitable compounds also include redoxcycling nitroxides such as TEMPOL, as well as targeted derivatives suchJP4-039 and the related family of compounds, and XJB-5-131 and therelated family of compounds. Suitable compounds also include thetranscription factor XBP1, its derivative XBP1s, and syntheticderivatives of XBP1 including XBP1 pathway stimulating factors. Suitablecompounds also include neurokin 1 receptor (NK1R) agonists includingtachykinins (e.g. substance P) and NKR1 such as aprepitant (Emend),their derivatives. A non-limiting listing of agents includesanti-Angiogenesis agents, anti-depressants, antidiabetic agents,antihistamines, anti-inflammatory agents, butorphanol, calcitonin andanalogs, COX-II inhibitors, dermatological agents, dopamine agonists andantagonists, enkephalins and other opioid peptides, epidermal growthfactors, erythropoietin and analogs, follicle stimulating hormone,glucagon, growth hormone and analogs (including growth hormone releasinghormone), growth hormone antagonists, heparin, hirudin and hirudinanalogs such as hirulog, IgE suppressors and other protein inhibitors,immunosuppressives, insulin, insulinotropin and analogs, interferons,interleukins, leutenizing hormone, leutenizing hormone releasing hormoneand analogs, monoclonal or polyclonal antibodies, motion sicknesspreparations, muscle relaxants, narcotic analgesics, nicotine,non-steroid anti-inflammatory agents, oligosaccharides, parathyroidhormone and analogs, parathyroid hormone antagonists, prostaglandinantagonists, prostaglandins, scopolamine, sedatives, serotonin agonistsand antagonists, tissue plasminogen activators, tranquilizers, vaccineswith or without carriers/adjuvants, vasodilators, major diagnostics suchas tuberculin and other hypersensitivity agents. Vaccine formulationsmay include an antigen or antigenic composition capable of eliciting animmune response against a human pathogen or from other viral pathogens.

Doxorubicin in Combination with Other Bioactive Components

In addition, as discussed in more detail below, each of the abovebioactive components can be integrated into a biocompatible material incombination with doxorubicin. Thus, for example, doxorubicin can be usedin combination with any of the other cytotoxic agents listed above(e.g., doxorubicin+cyclophosphamide, doxorubicin+5-fluorouracial, and soon) for using the delivery systems disclosed herein. When used incombination with doxorubicin, both the doxorubicin and the otherbioactive component can be linked to the CMC or other biocompatiblestructure. In another embodiment, one of the two components (eitherdoxorubicin or the other bioactive component) can be linked to the CMCor other biocompatible structural substrate as discussed herein and thesecond component (the remaining component) can be mixed (i.e., notgenerally chemically linked to the CMC or other biocompatible structure)into the biocompatible structure. In a third embodiment, as discussedelsewhere herein, both the doxorubicin and other bioactive component canbe freely integrated into the biocompatible structure.

In addition, as discussed in detail above, dissolvable microneedlearrays can also include doxorubicin in combination with each of theother bioactive agents described herein, including, for example,immunostimulants such as Poly(I:C). For example, as described herein,doxorubicin alone, Poly(I:C) alone, or doxorubicin and Poly(I:C) can beincorporated into the same MNA.

Linkage Strategies for Use with Bioactive Components and Substrates

In some embodiments, a linkage between the bioactive component(s) andthe substrate can be provided by a linker that is selected from thegroup consisting of a hydrazine group, a polypeptide, a disulfide group,and a thioether group.

As used herein, “linker” refers to a moiety that connects a first regionof the bioactive component to a second region of a biocompatiblematerial through chemical bonds (directly or indirectly). As describedherein, the connection can be severed so as to release a biologicallyactive form of the bioactive component. An example of a linker is amoiety that comprises a bond that is stable at neutral pH but is readilycleaved under conditions of low pH. Some linkers described herein can bemoieties that comprise a bond that is stable at pH values between 7 and8 but is readily cleaved at pH values between 4 and 6. Another exampleof a linker is a moiety that comprises a bond that is readily cleaved inthe presence of an enzyme. Preferred examples of such enzyme-sensitivelinkers are peptides comprising a recognition sequence for an endosomalpeptidase. Another example of a linker is a redox potential-sensitivelinker that is stable under conditions of low reduction potential (e.g.,low thiol or glutathione concentration) but cleaved under conditions ofhigh reduction potential (e.g., high thiol or glutathioneconcentration). Preferred examples of such redox potential-sensitivelinkers include disulfides and sulfenamides. Particularly preferredexamples include substituted aryl-alkyl disulfides in which the arylgroup is substituted with sterically-demanding and electron-withdrawingor electron-donating substitutents, so as to control the sensitivity ofthe disulfide linkage towards reaction with thiol. Another example of alinker is a moiety that comprises a bond that is readily cleaved uponexposure to radiation. Examples of such radiation-sensitive linkers are2-nitrobenzyl ethers that are cleaved upon exposure to light.Particularly preferred examples of linkers are moieties that mask thebiological activity of one of the two linked molecules until the linkageis severed.

In some embodiments, the linkage can be cleavable by a cleaving agentthat is present in the intracellular environment (e.g., within alysosome or endosome or caveolea). The linker can be, e.g., a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease.Typically, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells.Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in targeted cells or tissues. For example, a peptidyl linkerthat is cleavable by the thiol-dependent protease cathepsin-B, which ishighly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or aGly-Phe-Leu-Gly) linker). Other such linkers are described, e.g., inU.S. Pat. No. 6,214,345. In some embodiments, the peptidyl linkercleavable by an intracellular protease is a Val-Cit linker or a Phe-Lyslinker (see, e.g., U.S. Pat. No. 6,214,345, which describes thesynthesis of doxorubicin with the val-cit linker). One advantage ofusing intracellular proteolytic release of the therapeutic agent is thatthe agent is typically attenuated when conjugated and the serumstabilities of the conjugates are typically high.

In some embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929.) Such linkers are relativelystable under neutral pH conditions, such as those in the blood, but areunstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. Incertain embodiments, the hydrolyzable linker is a thioether linker (suchas, e.g., a thioether attached to the therapeutic agent via anacylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. See, e.g., U.S. Pat. No. 4,880,935.

In view of the many possible embodiments to which the principles of thedisclosed embodiments may be applied, it should be recognized that theillustrated embodiments are only preferred examples and should not betaken as limiting the scope of protection. Rather, the scope of theprotection is defined by the following claims. We therefore claim allthat comes within the scope and spirit of these claims.

We claim:
 1. A dissolvable microneedle array for transdermal insertioninto a patient comprising: a substrate comprising a biocompatiblematerial that forms base portion and a plurality of microneedlesextending from the base portion; and one or more bioactive componentsconjugated to the biocompatible material, wherein the one or morebioactive components are cleavable in vivo by an enzyme to release thebioactive component from the biocompatible material, or the bioactivecomponent retains function when conjugated to the biocompatiblematerial.
 2. The microneedle array of claim 1, wherein the biocompatiblematerial is carboxymethylcellulose.
 3. The microneedle array of claim 1,wherein the one or more bioactive components are bonded to thebiocompatible material by a disulfide bond.
 4. The microneedle array ofclaim 1, wherein the one or more bioactive components are cleavable invivo in response to pH, temperature, or both.
 5. The microneedle arrayof claim 1, wherein the one or more bioactive components compriseDoxorubicin.
 6. The microneedle array of claim 5, wherein the amount ofDoxorubicin ranges from about 1 to 1000 μg for chemotherapy.
 7. Themicroneedle array of claim 1, wherein the one or more bioactivecomponents are in a higher concentration in the plurality ofmicroneedles than in the base portion.
 8. The microneedle array of claim7, wherein substantially all of the one or more bioactive components arelocated in the plurality of microneedles so that the base portion issubstantially formed without any bioactive components contained therein,the one or more bioactive components are locally concentrated in theplurality of microneedles so that the one or more bioactive componentsare generally present only in an upper half of respective microneedlesin the microneedle array, and each microneedle comprises a plurality oflayers of the biocompatible material.
 9. The microneedle array of claim1, wherein the one or more bioactive components comprise at least twodifferent bioactive components conjugated to the biocompatible material.10. The microneedle array of claim 9, wherein the at least two differentbioactive components are selected from the group consisting of achemotherapeutic agent, an adjuvant, and a chemo attractant for a cancerchemo immunotherapy application.
 11. The microneedle array of claim 9,wherein the bioactive component comprises an antigen and an adjuvant fora vaccine application.
 12. The microneedle array of claim 1, wherein theone or more bioactive component comprises at least one viral vector. 13.The microneedle array of claim 12, wherein the at least one viral vectorcomprises an adenovector.
 14. A dissolvable microneedle array fortransdermal insertion into a patient comprising: a substrate comprisinga biocompatible material that forms base portion and a plurality ofmicroneedles extending from the base portion; and a first bioactivecomponent conjugated to the biocompatible material, the bioactivecomponent being Doxorubicin, wherein the first bioactive component iscleavable in vivo by an enzyme to release the first bioactive componentfrom the biocompatible material, or the first bioactive componentretains function when conjugated to the biocompatible material.
 15. Themicroneedle array of claim 14, wherein the biocompatible material iscarboxymethylcellulose.
 16. The microneedle array of claim 14, whereinthe first bioactive component is bonded to the biocompatible material bya disulfide bond.
 17. The microneedle array of claim 14, wherein thefirst bioactive components is cleavable in vivo in response to pH,temperature, or both.
 18. The microneedle array of claim 14, wherein theamount of Doxorubicin ranges from about 1 to 1000 μg for chemotherapy.19. The microneedle array of claim 14, further comprising a secondbioactive component.
 20. The microneedle array of claim 19, wherein thesecond bioactive component is selected from the group consisting ofPoly-IC or Poly-ICLC.
 21. The microneedle array of claim 19, wherein thesecond bioactive component is also conjugated to the biocompatiblematerial.
 22. The microneedle array of claim 19, wherein the secondbioactive component is mixed into the biocompatible material.
 23. Adissolvable microneedle array for transdermal insertion into a patientcomprising: a substrate comprising a biocompatible material that formsbase portion and a plurality of microneedles extending from the baseportion; and one or more bioactive components conjugated to thebiocompatible material, wherein the one or more bioactive components arecleavable in vivo to release the bioactive component from thebiocompatible material, or the bioactive component retains function whenconjugated to the biocompatible material, and wherein the one or morebioactive components are bonded to the biocompatible material by adisulfide bond which is cleavable in vivo by an enzyme.
 24. Themicroneedle array of claim 23, wherein the one or more bioactivecomponents are covalently bonded to the biocompatible material.
 25. Themicroneedle array of claim 23, wherein the one or more bioactivecomponents are bonded to the biocompatible material by a disulfide bond.26. The microneedle array of claim 23, wherein the biocompatiblematerial is carboxymethylcellulose.
 27. The microneedle array of claim23, wherein the one or more bioactive components comprise Doxorubicin.28. The microneedle array of claim 27, wherein the amount of Doxorubicinranges from about 1 to 1000 μg for chemotherapy.
 29. The microneedlearray of claim 23, wherein the one or more bioactive components are in ahigher concentration in the plurality of microneedles than in the baseportion.
 30. The microneedle array of claim 29, wherein substantiallyall of the one or more bioactive components are located in the pluralityof microneedles so that the base portion is substantially formed withoutany bioactive components contained therein, the one or more bioactivecomponents are locally concentrated in the plurality of microneedles sothat the one or more bioactive components are generally present only inan upper half of respective microneedles in the microneedle array, andeach microneedle comprises a plurality of layers of the biocompatiblematerial.
 31. The microneedle array of claim 23, wherein the one or morebioactive components comprise at least two different bioactivecomponents conjugated to the biocompatible material.
 32. The microneedlearray of claim 31, wherein the at least two different bioactivecomponents are selected from the group consisting of a chemotherapeuticagent, an adjuvant, and a chemo attractant for a cancer chemoimmunotherapy application.
 33. The microneedle array of claim 31,wherein the bioactive component comprises an antigen and an adjuvant fora vaccine application.
 34. The microneedle array of claim 23, whereinthe one or more bioactive component comprises at least one viral vector.35. The microneedle array of claim 34, wherein the at least one viralvector comprises an adenovector.
 36. A dissolvable microneedle array fortransdermal insertion into a patient comprising: a substrate comprisinga biocompatible material that forms base portion and a plurality ofmicroneedles extending from the base portion; and one or more bioactivecomponents conjugated to the biocompatible material, wherein the one ormore bioactive components are cleavable in vivo by an enzyme to releasethe bioactive component from the biocompatible material, or thebioactive component retains function when conjugated to thebiocompatible material, and wherein the one or more bioactive componentcomprises at least one viral vector wherein the bioactive component isbonded to the biocompatible material by a disulfide bond which iscleavable in vivo by an enzyme.
 37. The microneedle array of claim 36,wherein the one or more bioactive components are covalently bonded tothe biocompatible material.
 38. The microneedle array of claim 36,wherein the one or more bioactive components are bonded to thebiocompatible material by a disulfide bond.
 39. The microneedle array ofclaim 36, wherein the biocompatible material is carboxymethylcellulose.40. The microneedle array of claim 36, wherein the one or more bioactivecomponents are cleavable in vivo in response to pH, temperature, orboth.
 41. The microneedle array of claim 36, wherein the one or morebioactive components are in a higher concentration in the plurality ofmicroneedles than in the base portion.
 42. The microneedle array ofclaim 41, wherein substantially all of the one or more bioactivecomponents are located in the plurality of microneedles so that the baseportion is substantially formed without any bioactive componentscontained therein, the one or more bioactive components are locallyconcentrated in the plurality of microneedles so that the one or morebioactive components are generally present only in an upper half ofrespective microneedles in the microneedle array, and each microneedlecomprises a plurality of layers of the biocompatible material.
 43. Themicroneedle array of claim 36, wherein the one or more bioactivecomponents comprise at least two different bioactive componentsconjugated to the biocompatible material.
 44. The microneedle array ofclaim 43, wherein the bioactive component comprises an antigen and anadjuvant for a vaccine application.
 45. The microneedle array of claim36, wherein the at least one viral vector comprises an adenovector. 46.A dissolvable microneedle array for transdermal insertion into a patientcomprising: a substrate comprising a biocompatible material that formsbase portion and a plurality of microneedles extending from the baseportion; and one or more bioactive components conjugated to thebiocompatible material, wherein the one or more bioactive components arecleavable in vivo to release the bioactive component from thebiocompatible material, or the bioactive component retains function whenconjugated to the biocompatible material, wherein the one or morebioactive components comprise at least two different bioactivecomponents conjugated to the biocompatible material, and wherein thebioactive component comprises an antigen and an adjuvant for a vaccineapplication.
 47. The microneedle array of claim 46, wherein the one ormore bioactive components are covalently bonded to the biocompatiblematerial.
 48. The microneedle array of claim 46, wherein the one or morebioactive components are bonded to the biocompatible material by adisulfide bond.
 49. The microneedle array of claim 46, wherein thebiocompatible material is carboxymethylcellulose.
 50. The microneedlearray of claim 46, wherein the one or more bioactive components arecleavable in vivo by an enzyme.
 51. The microneedle array of claim 46,wherein the one or more bioactive components are cleavable in vivo inresponse to pH, temperature, or both.
 52. The microneedle array of claim46, wherein the one or more bioactive components further compriseDoxorubicin.
 53. The microneedle array of claim 52, wherein the amountof Doxorubicin ranges from about 1 to 1000 μg for chemotherapy.
 54. Themicroneedle array of claim 46, wherein the one or more bioactivecomponents are in a higher concentration in the plurality ofmicroneedles than in the base portion.
 55. The microneedle array ofclaim 54, wherein substantially all of the one or more bioactivecomponents are located in the plurality of microneedles so that the baseportion is substantially formed without any bioactive componentscontained therein, the one or more bioactive components are locallyconcentrated in the plurality of microneedles so that the one or morebioactive components are generally present only in an upper half ofrespective microneedles in the microneedle array, and each microneedlecomprises a plurality of layers of the biocompatible material.
 56. Themicroneedle array of claim 46, wherein the one or more bioactivecomponent further comprises at least one viral vector.
 57. Themicroneedle array of claim 56, wherein the at least one viral vectorcomprises an adenovector.
 58. A dissolvable microneedle array fortransdermal insertion into a patient comprising: a substrate comprisinga biocompatible material that forms base portion and a plurality ofmicroneedles extending from the base portion; and a first bioactivecomponent conjugated to the biocompatible material, the bioactivecomponent being Doxorubicin, wherein the first bioactive component iscleavable in vivo to release the first bioactive component from thebiocompatible material, or the first bioactive component retainsfunction when conjugated to the biocompatible material, and wherein thesecond bioactive component is mixed into the biocompatible materialwherein the bioactive components are bonded to the biocompatiblematerial by a disulfide bond which is cleavable in vivo by an enzyme.59. The microneedle array of claim 58, wherein the first bioactivecomponent is covalently bonded to the biocompatible material.
 60. Themicroneedle array of claim 58, wherein the first bioactive component isbonded to the biocompatible material by a disulfide bond.
 61. Themicroneedle array of claim 58, wherein the biocompatible material iscarboxymethylcellulose.
 62. The microneedle array of claim 58, whereinthe first bioactive components is cleavable in vivo in response to pH,temperature, or both.
 63. The microneedle array of claim 58, wherein theamount of Doxorubicin ranges from about 1 to 1000 μg for chemotherapy.64. The microneedle array of claim 58, wherein the second bioactivecomponent is selected from the group consisting of Poly-IC or Poly-ICLC.